Fusion protein including of CD4

ABSTRACT

Novel recombinant polypeptides are disclosed herein that include a CD4 polypeptide ligated at its C-terminus with a portion of an immunoglobulin comprising a hinge region and a constant domain of a mammalian immunoglobulin heavy chain. The portion or the IgG is fused at its C-terminus with a polypeptide comprising a tailpiece from the C-terminus of the heavy chain of an IgA antibody ara tailpiece from a C-terminus of the heavy chain of an IgM antibody. Also disclosed herein are methods for using these CD4 fusion proteins.

PRIORITY CLAIM

This is the § 371 U.S. National Stage of International Application No.PCT/US02/34393. filed Oct. 24. 2002, which was oublished in Englishunder PCT Article 21(2). which in turn claims the benefit of U.S.Provisional Patent Application No. 60/346,231, filed Oct. 25, 2001,incorporated by reference herein in its entirety.

FIELD OF THE INVENTION

This invention relates to the field of CD4 polypeptides, specifically toCD4 fusion proteins of use in the treatment of an immunodeficiency virusinfection such as a human immunodeficiency virus (HIV).

BACKGROUND OF THE INVENTION

The primary immunologic abnormality resulting from infection by HIV isthe progressive depletion and functional impairment of T lymphocytesexpressing the CD4 cell surface glycoprotein (Lane et al., Ann. Rev.Immunol. 3:477, 1985). CD4 is a non-polymorphic glycoprotein withhomology to the immunoglobulin gene superfamily (Maddon et al., Cell42:93, 1985). Together with the CD8 surface antigen, CD4 defines twodistinct subsets of mature peripheral T cells (Reinherz et al., Cell19:821, 1980), which are distinguished by their ability to interact withnominal antigen targets in the context of class I and class II majorhistocompatibility complex (MHC) antigens, respectively (Swain, Proc.Natl. Acad. Sci. 78:7101, 1981; Engleman et al., J. Immunol. 127:2124,1981; Spitz et al., J. Immunol. 129:1563, 1982; Biddison et al., J. Exp.Med. 156:1065, 1982; and Wilde et al., J. Immunol. 131:2178, 1983). Forthe most part, CD4 T cells display the helper/inducer T cell phenotype(Reinherz, supra), although CD4 T cells characterized ascytotoxic/suppressor T cells have also been identified (Thomas et al.,J. Exp. Med. 154:459, 1981; Meuer et al., Proc. Natl. Acad. Sci. USA79:4395, 1982; and Krensky et al., Proc. Natl. Acad. Sci. USA 79:2365,1982). The loss of CD4 helper/inducer T cell function probably underliesthe profound defects in cellular and humoral immunity leading to theopportunistic infections and malignancies characteristic of the acquiredimmunodeficiency syndrome (AIDS) (H. Lane supra).

Studies of HIV-I infection of fractionated CD4 and CD8 T cells fromnormal donors and AIDS patients have revealed that depletion of CD4 Tcells results from the ability of HIV-I to selectively infect, replicatein, and ultimately destroy this T lymphocyte subset (Klatzmann et al.,Science 225:59, 1984). The possibility that CD4 itself is an essentialcomponent of the cellular receptor for HIV-I was first indicated by theobservation that monoclonal antibodies directed against CD4 block HIV-Iinfection and syncytia induction (Dalgleish et al., Nature (London)312:767,1984; McDougal et al., J. Immunol. 135:3151, 1985). Thishypothesis has been confirmed by the demonstration that a molecularcomplex forms between CD4 and gp120, the major envelope glycoprotein ofHIV-I (McDougal et al., Science 231:382, 1986); and the finding thatHIV-I tropism can be conferred upon ordinarily non-permissive humancells following the stable expression of a CD4 cDNA (Maddon et al., Cell47:333, 1986).

The widespread use of highly active antiretroviral therapy (HAART) hasdramatically improved the clinical course for many individuals infectedwith HIV (Berrey, M. M. et al., J Infect Dis 183(10):1466, 2001).However, toxicities associated with long term HAART have put a highpriority on the design and development of less toxic therapies. Amongthe “next generation” of antiviral inhibitors is T-20 (Wild, C. et al.,Proc Natl Acad Sci USA 91(26):12676, 1994; Wild, et al. Proc Natl AcadSci USA 89(21):10537, 1992), a relatively non-toxic peptide thatdisrupts viral fusion thereby protecting CD4+ lymphocytes from de novoinfection. In clinical trials T-20 has been shown to reduce plasma viralload by up to two logs (Kilby, et al., Nat Med 4(11):1302, 1998). Theseresults demonstrate that the entry stage of the HIV replication cycle isa viable target for the development of new antiretroviral therapies.

Viral entry is a complex biochemical event that can be subdivided intoat least three stages: receptor docking, viral-cell membrane fusion, andparticle uptake (D'Souza, M.P. et al., Jama 284(2):215, 2000). Receptordocking is a multi-step process that begins with the gp120 component ofa virion spike binding to the CD4 receptor on the target cell.Conformational changes in gp120 induced by gp120-CD4 interaction promotea high affinity interaction between gp120 and either CCR5 or CXCR4cellular co-receptors. This is followed by gp41 mediated fusion of theviral and target cell membranes. Agents designed to block gp120-CD4,gp120-CCR5/CXCR4 or gp41/cell membrane interactions are in variousstages of development (D'Souza, M. P. et al., Jama 284(2):215, 2000).Several laboratories have constructed recombinant fusion proteins thatfuse the gp120 binding domain of CD4 to immunoglobulin constant domains(Deen, K. C. et al., Nature 331(6151):82, 1988; Fisher, R. A. et al.,Nature 331(6151):76, 1988; Capon, D. J. et al., Nature 337(6207):525,1989; Traunecker, A. et al., Nature 339(6219):68, 1989; Trkola, A. etal., J Virol 69(11):6609, 1995). One of these, Pro-542 is currentlybeing evaluated in clinical trials (Jacobson, J. M. et al., J Infect Dis182(1):326, 2000).

The strategy underlying these CD4 based therapies, i.e. blocking theinteraction between gp120 and the CD4 receptor, encompasses advantagesdistinct from current HAART regimens. The CD4 binding site on gp120includes highly conserved residues; thus, agents targeting this site areunlikely to encounter resistance mutants. Additionally, such agents, byblocking de novo infection, may prevent the expansion of viralreservoirs.

Monomeric soluble CD4 (sCD4) was one of the first reagents in this groupto be tested clinically (Schooley et al., Ann Intern Med 112(4):247,1990). Unfortunately, sCD4 failed to demonstrate significant antiviralactivity in vivo (Schooley et al., Ann Intern Med 112(4):247, 1990).Among the problems inherent to sCD4 was its inability to efficientlyneutralize primary isolates of HIV. The differential capacity of sCD4 toneutralize tissue culture laboratory adapted (TCLA) strains versus manyprimary isolates is striking. In the initial report describing thisdifference, Ho and colleagues found that the concentrations of sCD4required to neutralize primary isolates were up to 1000-fold higher thanthose required to neutralize TCLA strains (Ashkenazi et al., Proc NatlAcad Sci USA 88(16):7056, 1991). Surprisingly, when the affinities ofsCD4 for soluble gp120s derived from TCLA and primary isolates weremeasured, no correlation between sCD4 neutralization and CD4:gp120affinity was observed (Ashkenazi et al., Proc. Natl. Acad. Sci. USA88(16):7056,1991; Brighty et al., Proc. Natl. Acad. Sci. USA88(17):7802, 1991; Ivey-Hoyle et al., Proc. Natl. Acad. Sci. USA88(2):512, 1991). However, the affinity of sCD4 for gp120 on primaryvirions was reduced relative to gp120 on the surface of TCLA virions(Moore et al., J Virol 66, 235-243, 1992). The basis for thedifferential interaction between sCD4 and soluble gp120 vs. virionassociated gp120 is unclear.

There is an additional property of sCD4 that may, at least in part,explain its inability to neutralize primary isolates. At lowconcentrations sCD4 enhances the infectivity of most primary isolates(Moore et al., Aids 9, Suppl A:S117, 1995; Sullivan et al., J Virol69(7):4413, 1995; Moore et al., J Virol 66(1):235, 1992; Orloff et al.,J Virol 67(3):1461, 1993; Schutten et al., Scand J Immunol 41(1):18,1995; Willey et al., J Virol 68(2):1029, 1994). Although theseobservations were made prior to the identification of the HIVfusion/coreceptors, several research groups suggested that sCD4 mediatedenhancement resulted from the activation of the fusion component ofvirion associated spikes (Sullivan et al., J Virol 69(7):4413, 1995; Fuet al., J Virol 67(7):3818, 1993). As has since become clear, sCD4engagement of gp120 results in conformational changes in gp120 thatpromote its interaction with CCR5 and thus initiates the process ofvirus-cell fusion (Doranz et al., J Virol 73(12):10346, 1999; Trkola etal., Nature 384(6605):184, 1996; Wu et al., Nature 384(6605):179, 1996;Zhang et al., Biochemistry 38(29):9405, 1999).

Because sCD4-mediated enhancement of virus infectivity is only observedat low concentrations of sCD4, it likely reflects a condition wherevirions bear a mixture of unoccupied gp120s along with sCD4-boundgp120s. Neutralization occurs only when the concentration of sCD4reaches a threshold level where a sufficient number of spikes per virionare prevented from participating in the fusion process. Theconcentration required to achieve that state is likely to be extremelyhigh for two reasons: 1) sCD4 must compete with surface bound CD4receptors which are presented in bulk on the surface of a target celland the effects of avidity strongly favor the receptors presented on themembrane. The lack of high avidity associated with monomeric sCD4 is acritical deficiency in the antiviral activity of this molecule, and 2)sCD4 promotes a high affinity interaction between gp120 and CCR5 (Doranzet al., J Virol 73(12):10346, 1999; Trkola, et al., Nature384(6605):184, 1996; Wu et al., Nature 384(6605):179, 1996; Zhang etal., Biochemistry 38(29):9405, 1999). Thus, even at relatively highconcentrations, sCD4 promotes interactions between the virion and thetarget cell membrane.

Regardless of the mechanism, it is clear that sCD4 is not thetherapeutic agent of choice for treating HIV. Thus, a need remains for aCD4-based agent that can be used to study HIV infection in vitro, and isof use for treating or preventing HIV infection in vivo.

SUMMARY OF THE INVENTION

Novel recombinant polypeptides are disclosed herein that include a CD4polypeptide ligated at its C-terminus with a portion of animmunoglobulin comprising a hinge region and a constant domain of amammalian immunoglobulin heavy chain. The portion of the IgG is fused atits C-terminus with a polypeptide comprising a tailpiece from the Cterminus of the heavy chain of an IgA antibody. Also disclosed hereinare methods for using these CD4-fusion proteins.

The foregoing and other objects, features, and advantages of theinvention will become more apparent from the following detaileddescription of several embodiments which proceeds with reference to theaccompanying figures.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1A is a digital image showing gel-filtration of purified D1D2-Igαtpand FIG. 1B is a digital image showing multiple recombinant HIV gp120proteins.

D1D2-Igαtp was purified from CHO culture supernatants and passed over aSuperdex-200 gel-filtration column at a flow rate of 0.5 ml/min.Absorbance was measured at 280 nm and 0.5 ml fractions were collected.Molecular weight standards were also run under the same conditions togenerate a standard curve (left inset). The void volume of this columnwas determined to be 7.45 mls. Peak fractions were collected andelectrophoresed through a denaturing SDS-polyacrylamide gel and analyzedby western blot with an anti-CD4 polyclonal antisera (right inset). sCD4was used as a positive control in the western blot. In FIG. 1B, acomplex of D1D2Igαtp and multiple subunits of gp120 are passed over asuperose-6 10/30 column at a flow rate of 0.3 ml/min. This chromatogramis compared chromatograms of D1D2Igαtp alone and gp120 alone. Thepresence of both D1D21gαtp and gp120 in the peak fraction is confirmedby western blot analysis (see insets).

FIG. 2 is a digital image showing a comparison of sCD4 and D1D2-Igαtp ina real-time PCR based viral entry assay. PBMCs were inoculated withHIV-1 JR-FL alone or in the presence of either sCD4 or D1D2-Igαtp. Thenumber of virions entering cells within 6 hrs. post infection wasdetermined using a real-time PCR based viral entry assay in whichearly-LTR reverse transcripts were enumerated. A standard curve wasgenerated from genomic DNA obtained from an ACH-2 cell line carrying asingle integrated HIV-1 genome. FIG. 2A shows a direct comparison ofsCD4 vs. D1D2-Igαtp. FIG. 2B is a further titration of D1D2-Igαtp. Thecorrelation coefficient for these two experiments was 0.993 and 0.998respectively with slopes of −3.209 and −3.356 respectively. Theseresults are representative of at least three independent experimentsusing different donor PBMCs.

FIG. 3 is a digital image of propagation of HIV-1 from infected patientPBMCs in the presence of sCD4 versus D1D2-Igαtp. PBMCs derived from HIV+patients were co-cultured with uninfected donor PBMCs and treated inparallel with either sCD4 or D1D2-Igαtp. Viral replication was measuredby p24 antigen ELISA in culture supernatants. Three different patientsamples were analyzed. Levels of p24 were measured on the day of peakreplication in cultures containing various concentrations (3 nM, 6 nM or12 nM) of either sCD4 or D1D2-Igαtp (FIGS. 3B, 3C, and 3D). A timecourse for donor 1, treated with 6 nM sCD4 or D1D2-Igαtp is shown inpanel a.

FIG. 4 are graphs of data obtained from acute infection of uninfectedPBMCs with HIV-1 Bal and a primary viral isolate in the presence ofsCD4, D1D2-Igαtp, and mAb 17b. Activated PBMCs from uninfected donorswere acutely infected with HIV-1 Bal (FIG. 4A) or a primary isolate(FIG. 4B) in the presence of sCD4, D1D2-Igαtp, mAb 17b and combinationsof sCD4 and 17b or D1D2-Igαtp and 17b. p24 values were measured everytwo days and values from the peak day of replication are reported. Theseresults are representative of at least three independent experimentsusing different donor PBMCs.

FIG. 5 are digital images showing biosensor analysis of thestoichiometry of gp120 monomers bound per D1D2-Igαtp. FIG. 5A is asensorgram overlay of increasing concentrations of NL-4-3 gp120 bindingto D1D2-Igαtp. D1D2-Igαtp 1 was bound to protein A, previouslyimmobilized to a density of 735 RU, starting at point (a) for a total of2 min, ending at point (b). After a 5 minute washout period to allowthis surface to stabilize, NL-4-3 gp120 was injected starting at point(c) at the concentrations shown in the inset, and ending at point (d).The amount of protein bound, in response units (RU), was determined atpoints (c) and (d) for D1D2-Igαtp and gp120, respectively, in eachcycle. In FIG. 5B the total mass of each protein bound was determined asdescribed in materials and methods and are presented as the ratio of thenumber of gp120 monomers bound per D1D2-Igαtp.

FIG. 6 are digital images that show the relative rates of dissociationof gp120 from sCD4 and D1D2-Igαtp. In FIG. 6A Soluble CD4 (sCD4) orD1D2-Igαtp was attached to a CM5 sensor surface either by direct aminecoupling (sCD4, 400-500 RU), or indirectly using protein A (D1D2-Igαtp,200-250 RU). The indicated gp120 s (100 nM each) were then passed overthe surfaces at 25 μl/min for 2 minutes at which point running buffer inthe absence of gp120 was passed over the surfaces to allow dissociationof bound proteins. As the association phase of each ligand-analyte pairshowed little variation in binding rates, only the dissociation phase ofeach sensorgram is shown. Each curve was normalized to account fordifferences in total response in the individual experiments. In FIG. 6Bgp120 was attached to the CM5 sensor chip by direct amine coupling, andJR-FL gp120 was passed over the chip at increasing concentrations.

FIG. 7 are digital images that show hydrodynamic and thermodynamicstudies of the size-distributions of D1D2-Igαtp. Sedimentationcoefficient distributions c(s) of the peak D1D2-Igαtp fraction (solidline) and trailing fraction (dashed line) are represented. The arrowsindicate the estimated range of sedimentation coefficients for thedifferent oligomers, which results in hydrodynamic radius values of11.9-12.9 nm for a 0pentamer (with 14-15 S), 12.7-13.5 nm for a hexamer(16-17 S), 12.6-13.3 nm for a heptamer (19-20 S), and 12.5-13.1 nm foran octamer (22-23 S). The top inset shows sedimentation equilibrium dataof D1D2-Igαtp at 3,000 rpm (squares), 5,000 rpm (circles), and 7,500 rpm(triangles), This resulted in 1,240 kDa (or 8.8 monomer units) at 3,000rpm to 954 kDa (6.8 units) at 5,000 rpm, and 810 kDa (5.8 units) at7,500 rpm. The bottom inset shows the hydrodynamic radius distributioncalculated from dynamic light scattering data, for the peak fraction(solid line) and trailing fraction (dashed line). The contribution tothe scattering intensity increases with size of the molecules, andtherefore overemphasizes the abundance of larger species in the peakfraction.

FIG. 8 is a graph showing the neutralization of four primary isolates ofHIV-1 by D1D2-Igαtp. Four minimally passaged primary isolates of HIV-1were preincubated with D1D2-Igαtp and then added to a culture ofactivated PBMCs. Cultures were maintained in standard culture media andneutralization assays were in a standard manner. reverse transcriptasepresent in the viral supernatant was measured for each day.Neutralization is reported as the percent inhibition relative to viruswithout any inhibitor, and reported just prior to the day of peakreplication.

FIG. 9 is a bar graph of viral entry assay in which D1D2-Igαtp was addedone or two hours after exposure of PBMC to HIV-1. Viral entry assayswere carried out as described in FIG. 2, however, D1D2-Igαtp was addedafter virus was allowed to attach to cells.

FIG. 10 is set of line graphs showing the binding of D1D2-Igαtp orFD1D2-Igαtp to cell expressing CD16 (FcγRIII) or CD32 (FcγRII). Theresults from competition experiments using a labeled anti CD16 or antiCD32 antibody as a competitor are shown. Results are expressed as theinhibition of binding of the antibody to either CD16 or CD32. FIG. 10Ashows the binding to CD16 obtained in the presence of 1-1000 nM ofcompetitor. D1D2-Igαtp efficiently competes for binding to CD16, whileFD1D2-Igαtp competes less efficiently. Antibody 2G12 (negative control,a human IgG₁) did not compete for binding to CD16. The % CD16 meanchannel fluorescence (mcf) was calculated as follows:

${\%\mspace{14mu}{CD16}\mspace{14mu}{mcf}} = {\frac{\begin{matrix}{\left( {{CD16mcf}\text{-}{backgroud}} \right) -} \\\left( {{CD16}\mspace{14mu}{with}\mspace{14mu}{inhibitor}\mspace{14mu}{mcf}\text{-}{background}} \right.\end{matrix}}{\left( {{CD16}\mspace{14mu}{mcf}\text{-}{background}} \right)} \times 100}$

FIG. 10B shows the binding to CD32 obtained in the presence of 1-1000 nMof competitor (2G12, a human IgG₁). D1D2-Igαtp efficiently competes forbinding to CD32, while FD1D2-Igαtp competes less efficiently. Antibody2G12, (negative control, a human IgG₁), did not compete for binding toCD32. The % CD32 mcf was calculated as follows:

${\%\mspace{14mu}{CD32}\mspace{14mu}{mcf}} = {\frac{\begin{matrix}{\left( {{CD32mcf}\text{-}{backgroud}} \right) -} \\\left( {{CD32}\mspace{14mu}{with}\mspace{14mu}{inhibitor}\mspace{14mu}{mcf}\text{-}{background}} \right.\end{matrix}}{\left( {{CD32}\mspace{14mu}{mcf}\text{-}{background}} \right)} \times 100}$

FIG. 11 is a series of plots showing the induction of a calcium flux byD1D2-Igαtp or FD1D2-Igαtp (mutant F) in natural killer (NK) cells afterthe cells were cultured in vitro for 14 days. Each point shownrepresents a single cell. The negative control (SHAM, FIG. 11A) did notexhibit any calcium influx, while application of differentconcentrations of D1D2-Igαtp (FIGS. 11B-F, 120 nM, 60 nM, 30 nm, 15 nM,and 7.5 nM, respectively) elicited a calcium influx. Mutant Fapplication did not induce a calcium influx (FIG. 11GK, 120 nM, 60 nM,30 nM, 15 nM, and 7.5 nM, respectively), indicated that this moleculedoes not activate natural killer cells.

FIG. 12 is a set of plots from Fluorescence Activated Cell SortingAnalyses demonstrating that natural killer cells in the presence ofD1D2-Igαtp mediate antibody dependent cell mediated cytoxicity. Naturalkiller (NK) cells and HIV-infected CEM.NRK target cells were incubatedin the presence of D1D2-Igαtp or in media alone. Cells were subsequentlylabeled with propidium iodide, which measures cell viability. In thepresence of D1D2-Igαtp, 45% of the HIV-1 infected cells were killed(FIG. 13A), whereas without application of D1D2-Igαtp, only 15% of thecells were killed (FIG. 13B). The same number of uninfected CEM.NRKcells survived in the presence of D1D2-Igαtp (FIG. 13D) as compared touninfected CEM.NRK cells incubated in the absence of antibody.

FIG. 13 is a line graph showing the percent of PI positive cellsobtained after incubation of HIV-infected CEM.NRK target cells with NKcells in the presence of either D1D2-Igαtp or FD1D2-Igαtp (labeled“mutant F”). Both D1D2-Igαtp and FD1D2-Igαtp induced killing, althoughD1D2-Igαtp was more effective.

FIG. 14 is a schematic diagram showing D1D2-Igαtp and the residuesaltered to obtain mutant F. In FD1D2-Igαtp, principle residues in thearea responsible for binding to Fc gamma receptors (bright areas ofresidues responsible for binding of the immunoglobulin molecule to Fc)are mutated. In FD1D2-Igαtp, amino acid residues 218-221 are replaced bythe corresponding residues of an IgG₂. Thus,218-Glu Leu Leu Gly Gly Pro-221(corresponding to residues 233-238 in an intact immunoglobulin molecule,using the numbering system of Kabat et al., “Sequences of proteins ofimmunological interest.” U.S. Department of Health and Human Services,National Institutes of Health, Bethesda, Md., 1991) is replaced by218-Pro Val—Ala Gly Pro-221.

It should be noted that replacement of one or more residues of Asn 297,Asp 265, P329, Asp 270, Ala 327, Ser 239, Lys 338 (using the numberingsystem of Kabat et al., “Sequences of proteins of immunologicalinterest.” U.S. Department of Health and Human Services, NationalInstitutes of Health, Bethesda, Md., 1991) (see Shields et al., J. BiolChem. 276(9): 6591-604, 2001) with other amino acids, will induce asimilar effect.

DETAILED DESCRIPTION OF SEVERAL EMBODIMENTS

The following definitions and methods are provided to better define thepresent invention and to guide those of ordinary skill in the art in thepractice of the present invention. It must be noted that as used hereinand in the appended claims, the singular forms “a”, “an”, and “the”include plural referents unless the context clearly dictates otherwise.Thus, for example, reference to “a cell” includes a plurality of suchcells and reference to “the antibody” includes reference to one or moreantibodies and equivalents thereof known to those skilled in the art,and so forth.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood to one of ordinary skill inthe art to which this invention belongs.

Term

Alpha tailpiece (αtp): The tailpiece located at the C-terminus of theheavy chain of an IgA antibody. In one embodiment, this peptide is 18amino acids in length and is derived from a human IgA molecule. In oneembodiment, an alpha tailpiece is PTHVNVSVVMAEVDGTCY (SEQ ID NO:1).However, if desired the peptide may be modified to remove theglycosylation site by changing 1 or 2 amino acids at residues 5-7 (NVS).For example, the asparagine (N) at position 5 can be changed to aglutamine (Q). Alternatively, the serine (S) at position 7 can bechanged to an alanine (A). Additionally, a few of the amino acidsresidues of the IgA constant region may also be included, such as aboutfour amino acids of the IgA constant region. Suitable IgA molecules,having an alpha tailpiece of use include, but are not limited to, humanIgA1, human IgA2, rabbit IgA, and mouse IgA. This peptide is linked,either directly or indirectly to a constant domain of an immunoglobulin,such as a fragment including the CH2 and CH3 domains.

Animal: A living multicellular vertebrate organism, a category whichincludes, for example, mammals and birds. A “mammal” includes both humanand non-human mammals. Similarly, the term “subject” includes both humanand veterinary subjects.

Antibody: Immunoglobulin molecules and immunologically active portionsof immunoglobulin molecules, i.e., molecules that contain an antigenbinding site which specifically binds (immunoreacts with) an antigen.

A naturally occurring antibody (e.g., IgG) includes four polypeptidechains, two heavy (H) chains and two light (L) chains inter-connected bydisulfide bonds. However, it has been shown that the antigen-bindingfunction of an antibody can be performed by fragments of a naturallyoccurring antibody. Thus, these antigen-binding fragments are alsointended to be designated by the term “antibody”. Examples of bindingfragments encompassed within the term antibody include (i) an Fabfragment consisting of the VL, VH, CL and CH1 domains; (ii) an Fdfragment consisting of the VH and CH1 domains; (iii) an Fv fragmentconsisting of the VL and VH domains of a single arm of an antibody, (iv)a dAb fragment (Ward et al., Nature 341:544-546, 1989) which consists ofa VH domain; (v) an isolated complimentarily determining region (CDR);and (vi) an F(ab′)₂ fragment, a bivalent fragment comprising two Fabfragments linked by a disulfide bridge at the hinge region.

Antigen Presenting Cell (APC): Cells that present antigen to the immunesystem. There are three general classes of antigen presenting cells(APCs): macrophages, dendritic cells, and B cells, although neutrophilscan also present antigens. Processing and surface presentation ofantigen by APCs can be thought of as a first step in the normal immuneresponse. The antigen can be any antigen, including but not limited toan antigen of a bacterial, virus, fungus, or any other infectiousorganism.

“Antigen presentation” is the set of events whereby cells fragmentantigens into peptides, and then present these peptides in associationwith products of the major histocompatibility complex, (MHC). The MHC isa region of highly polymorphic genes whose products are expressed on thesurfaces of a variety of cells. T cells recognize foreign antigens boundto only one specific class I or class II MHC molecule. Activated or“stimulated” antigen presenting cells are uniquely capable of processingand presenting antigens to naive T cells. The patterns of antigenassociation with either a class I or class II MHC molecule determineswhich T cells are stimulated.

Avidity: The overall strength of interaction between two molecules, suchas an antigen and an antibody. Avidity depends on both the affinity andthe valency of interactions. Therefore, the avidity of a pentameric IgMantibody, with ten antigen binding sites, for a multivalent antigen maybe much greater than the avidity of a dimeric IgG molecule for the sameantigen.

Binding or stable binding: An oligonucleotide binds or stably binds to atarget nucleic acid if a sufficient amount of the oligonucleotide formsbase pairs or is hybridized to its target nucleic acid, to permitdetection of that binding. Binding can be detected by either physical orfunctional properties of the target:oligonucleotide complex. Bindingbetween a target and an oligonucleotide can be detected by any procedureknown to one skilled in the art, including both functional and physicalbinding assays. Binding can be detected functionally by determiningwhether binding has an observable effect upon a biosynthetic processsuch as expression of a gene, DNA replication, transcription,translation and the like.

Physical methods of detecting the binding of complementary strands ofDNA or RNA are well known in the art, and include such methods as DNaseI or chemical footprinting, gel shift and affinity cleavage assays,Northern blotting, dot blotting and light absorption detectionprocedures. For example, one method that is widely used, because it isso simple and reliable, involves observing a change in light absorptionof a solution containing an oligonucleotide (or an analog) and a targetnucleic acid at 220 to 300 nm as the temperature is slowly increased. Ifthe oligonucleotide or analog has bound to its target, there is a suddenincrease in absorption at a characteristic temperature as theoligonucleotide (or analog) and target disassociate from each other, ormelt.

The binding between an oligomer and its target nucleic acid isfrequently characterized by the temperature (T_(m)) at which 50% of theoligomer is melted from its target. A higher (T_(m)) means a stronger ormore stable complex relative to a complex with a lower (T_(m)).

CD4: Cluster of differentiation factor 4 polypeptide, a T-cell surfaceprotein that mediates interaction with the MHC class II molecule. CD4also serves as the primary receptor site for HIV on T-cells during HIVinfection.

The known sequence of the CD4 precursor has a hydrophobic signalpeptide, an extracelluar region of approximately 370 amino acids, ahighly hydrophobic stretch with significant identify to themembrane-spanning domain of the class II MHC beta chain, and a highlycharged intracellular sequence of 40 resides (Maddon, Cell 42:93, 1985).

The term “CD4” includes polypeptide molecules that are derived from CD4include fragments of CD4, generated either by chemical (e.g., enzymatic)digestion or genetic engineering means. Such a fragment may be one ormore entire CD4 protein domains. The extracellular domain of CD4consists of four contiguous immunoglobulin-like regions (D1, D2, D3, andD4, see Sakihama et al., Proc. Natl. Acad. Sci. 92:6444, 1995; U.S. Pat.No. 6,117,655), and amino acids 1 to 183 have been shown to be involvedin gp120 binding. For instance, a binding molecule or binding domainderived from CD4 would comprise a sufficient portion of the CD4 proteinto mediate specific and functional interaction between the bindingfragment and a native or viral binding site of CD4. One such bindingfragment includes both the D1 and D2 extracellular domains of CD4 (D1D2is also a fragment of soluble CD4 or sCD4 which is comprised of D1 D2 D3and D4), although smaller fragments may also provide specific andfunctional CD4-like binding. The gp120-binding site has been mapped toD1 of CD4.

CD4 polypeptides also include “CD4-derived molecules” which encompassesanalogs (non-protein organic molecules), derivatives (chemicallyfunctionalized protein molecules obtained starting with the disclosedprotein sequences) or mimetics (three-dimensionally similar chemicals)of the native CD4 structure, as well as proteins sequence variants orgenetic alleles, that maintain the ability to functionally bind to atarget molecule.

cDNA (complementary DNA): A piece of DNA lacking internal, non-codingsegments (introns) and transcriptional regulatory sequences. cDNA canalso contain untranslated regions (UTRs) that are responsible fortranslational control in the corresponding RNA molecule. cDNA issynthesized in the laboratory by reverse transcription from messengerRNA extracted from cells.

Constant domain of an immunoglobulin heavy chain or Fc: There is aregion of about 110 amino acids in the N-terminal portion of theheavy-chain which always differs in amino acid sequence from one Igheavy-chain preparation to another. The remainder of the heavy-chainsequence shows little if any amino acid-sequence difference, and arecalled the constant or “C” regions. These C-region sequences can begrouped into 5 classes of immunoglobulin heavy-chains named: gamma, mu,alpha, delta, and epsilon. The immunoglobulins from which these heavychains are obtained were are named: IgG, IgM, IgA, IgD, and IgE,respectively. The heavy chains of IgG, IgA, and IgD are about 50,000 MW,while the heavy chains of IgM and IgE are about 65,000 MW. For anindividual immunoglobulin molecule there are at least two heavy-chainsof identical sequence on a given molecule (analogous to the constantregion of light chains on a given molecule). In addition, the pattern of110 amino-acid-in length segments was retained in each heavy chain.Thus, IgG heavy chain has about 110 amino acids in the N-terminalportion, then a 110 amino acid segment designated CH1 (constant heavy1), then a 110 amino acid segment named CH2, then finally, a 110 aminoacid segment named CH3. IgG, IgA and IgE each include 3 constant domainsnamed CH1, CH2, and CH3, while IgM includes CH1, CH2, CH3, and aadditional CH4 region. As the amino acid sequence of the differentheavy-chain classes differ significantly within a species they can bereadily distinguished from one another. A “hinge” region of animmunoglobulin is an amino acid sequence that connects CH2 and CH3 toeach other. In one embodiment, an immunoglobulin Fc includes the CH2 andCH3 regions, and can also include the hinge region.

Conservative substitutions: Modifications of a polypeptide that involvethe substitution of one or more amino acids for amino acids havingsimilar biochemical properties that do not result in change or loss of abiological or biochemical function of the polypeptide. These“conservative substitutions” are likely to have minimal impact on theactivity of the resultant protein. Table 1 shows amino acids that may besubstituted for an original amino acid in a protein, and which areregarded as conservative substitutions.

TABLE 1 Original Residue Conservative Substitutions Ala ser Arg lys Asngln; his Asp glu Cys ser Gln asn Glu asp Gly pro His asn; gln Ile leu;val Leu ile; val Lys arg; gln; glu Met leu; ile Phe met; leu; tyr Serthr Thr ser Trp tyr Tyr trp; phe Val ile; leu

In one embodiment, one or more conservative changes, or up to tenconservative changes, can be made in a polypeptide without changing abiochemical function of the polypeptide. For example, one or moreconservative changes can be made in a CD4 D1D2 polypeptide withoutchanging its ability to bind to gp120. More substantial changes in abiochemical function or other protein features may be obtained byselecting amino acid substitutions that are less conservative than thoselisted in Table 2. Such changes include, for example, changing residuesthat differ more significantly in their effect on maintainingpolypeptide backbone structure (e.g., sheet or helical conformation)near the substitution, charge or hydrophobicity of the molecule at thetarget site, or bulk of a specific side chain. The followingsubstitutions are generally expected to produce the greatest changes inprotein properties: (a) a hydrophilic residue (e.g., seryl or threonyl)is substituted for (or by) a hydrophobic residue (e.g., leucyl,isoleucyl, phenylalanyl, valyl or alanyl); (b) a cysteine or proline issubstituted for (or by) any other residue; (c) a residue having anelectropositive side chain (e.g., lysyl, arginyl, or histadyl) issubstituted for (or by) an electronegative residue (e.g., glutamyl oraspartyl); or (d) a residue having a bulky side chain (e.g.,phenylalanine) is substituted for (or by) one lacking a side chain(e.g., glycine).

DNA: Deoxyribonucleic acid. DNA is a long chain polymer which comprisesthe genetic material of most living organisms (some viruses have genescomprising ribonucleic acid (RNA)). The repeating units in DNA polymersare four different nucleotides, each of which comprises one of the fourbases, adenine, guanine, cytosine and thymine bound to a deoxyribosesugar to which a phosphate group is attached. Triplets of nucleotides(referred to as codons) code for each amino acid in a polypeptide. Theterm codon is also used for the corresponding (and complementary)sequences of three nucleotides in the mRNA into which the DNA sequenceis transcribed.

Deletion: The removal of a sequence of DNA, the regions on either sidebeing joined together.

Encode: A polynucleotide is said to “encode” a polypeptide if, in itsnative state or when manipulated by methods well known to those skilledin the art, it can be transcribed and/or translated to produce the mRNAfor and/or the polypeptide or a fragment thereof. The anti-sense strandis the complement of such a nucleic acid, and the encoding sequence canbe deduced therefrom.

Functional fragments and variants of a polypeptide: Includes thosefragments and variants that maintain one or more functions of the parentpolypeptide. It is recognized that the gene or cDNA encoding apolypeptide can be considerably mutated without materially altering oneor more the polypeptide's functions. First, the genetic code is wellknown to be degenerate, and thus different codons encode the same aminoacids. Second, even where an amino acid substitution is introduced, themutation can be conservative and have no material impact on theessential functions of a protein. See Stryer, Biochemistry 3rd Ed.,1988. Third, part of a polypeptide chain can be deleted withoutimpairing or eliminating all of its functions. Fourth, insertions oradditions can be made in the polypeptide chain, for example, addingepitope tags—without impairing or eliminating its functions (Ausubel etal., J. Immunol. 159:2502, 1997). Other modifications that can be madewithout materially impairing one or more functions of a polypeptideinclude, for example, in vivo or in vitro chemical and biochemicalmodifications or which incorporate unusual amino acids. Suchmodifications include, for example, acetylation, carboxylation,phosphorylation, glycosylation, ubiquination, labeling, e.g., withradionuclides, and various enzymatic modifications, as will be readilyappreciated by those well skilled in the art. A variety of methods forlabeling polypeptides and of substituents or labels useful for suchpurposes are well known in the art, and include radioactive isotopessuch as ³²P, ligands which bind to labeled antiligands (e.g.,antibodies), fluorophores, chemiluminescent agents, enzymes, andantiligands. Functional fragments and variants can be of varying length.For example, some fragments have at least 10, 25, 50, 75, 100, or 200amino acid residues.

A functional fragment or variant of CD4 is defined herein as apolypeptide which binds to gp120. It includes any polypeptide six ormore amino acid residues in length which is capable of binding gp120, orbinds an MCH class II molecule.

Gp120: The envelope protein from Human Immunodeficiency Virus (HIV). Theenvelope protein is initially synthesized as a longer precursor proteinof 845-870 amino acids in size, designated gp160. Gp160 forms ahomotrimer and undergoes glycosylation within the Golgi apparatus. It isthen cleaved by a cellular protease into gp120 and gp41. Gp41contains atransmembrane domain and remains in a trimeric configuration; itinteracts with gp120 in a non-covalent manner. Gp120 contains most ofthe external, surface-exposed, domains of the envelope glycoproteincomplex, and it is gp120 which binds both to the cellular CD4 receptorand to the cellular chemokine receptors (e.g., CCR5)

The gp120 core has a unique molecular structure, that comprises twodomains: an “inner” domain (which faces gp41) and an “outer” domain(which is mostly exposed on the surface of the oligomeric envelopeglycoprotein complex). The two gp120 domains are separated by a“bridging sheet” that is not part of either domain. Binding to CD4causes a conformational change in gp120 which exposes the bridging sheetand may move the inner and outer domains relative to each other. TheCD4-binding pocket within gp120 comprises a number of residues whichinteract directly with Phe43 of CD4. The most important of these areGlu370, Trp427 and Asp368 (the latter residue also forms a salt bridgewith Arg59 of CD4). These three residues are conserved in all primatelentiviruses.

Immunoglobulins: A class of proteins found in plasma and other bodyfluids that exhibits antibody activity and binds with other moleculeswith a high degree of specificity; divided into five classes (IgM, IgG,IgA, IgD, and IgE) on the basis of structure and biological activity.Immunoglobulins and certain variants thereof are known and many havebeen prepared in recombinant cell culture (e.g., see U.S. Pat. Nos.4,745,055; 4,444,487; WO 88/03565; EP 256,654; EP 120,694; EP 125, 023;Faoulkner et al., Nature 298:286, 1982; Morrison, J. Immunol. 123:793,1979; Morrison et al., Ann Rev. Immunol. 2:239, 1984). A chimericimmunoglobulin includes residues primarily from a first class ofimmunoglobulin, with amino acids substituted by the correspondingresidues of a second class of immunoglobulin. In one specific,non-limiting example, a chimeric IgG₁ includes corresponding residuesfrom an IgG₂. In several non-limiting examples, at least one, about oneto about twenty, about one to about ten, about one to about five, orabout four residues are substituted.

A native (naturally occurring) immunoglobulin is each is made up of fourpolypeptide chains. There are two long chains, called the “heavy” or “H”chains which weigh between 50 and 75 kilodaltons and two short chainscalled “light” or “L” chains weighing in at 25 kilodaltons. They arelinked together by what are called disulfide bonds to form a “Y” shapemolecule. Each heavy chain and light chain can be divided into avariable region and a constant region. An Fc region includes theconstant regions of the heavy and the light chains, but not the variableregions. Fc receptors are those receptors that specifically bind an Fcregion of an immunoglobulin. These receptors include, but are notlimited to, FcαRII, FcαRIII, and FcRN.

Isolated: An “isolated” biological component (such as a nucleic acidmolecule, protein or organelle) has been substantially separated orpurified away from other biological components in the cell of theorganism in which the component naturally occurs, i.e., otherchromosomal and extra-chromosomal DNA and RNA, proteins and organelles.Nucleic acids and proteins that have been “isolated” include nucleicacids and proteins purified by standard purification methods. The termalso embraces nucleic acids and proteins prepared by recombinantexpression in a host cell as well as chemically synthesized nucleicacids.

Lymphocytes: A type of white blood cell that is involved in the immunedefenses of the body. There are two main types of lymphocytes: B-cellsand T-cells.

Mu tailpiece (μtp): The tailpiece located at the C-terminus of the heavychain of an IgM antibody. In one embodiment, this peptide is 18 aminoacids in length and is derived from an IgM molecule.

Natural Killer Cell: A large granular lymphocyte capable of killing atumor or microbial cell without prior exposure to the target cell andwithout having it presented with or marked by a histocompatibilityantigen.

Nucleotide: “Nucleotide” includes, but is not limited to, a monomer thatincludes a base linked to a sugar, such as a pyrimidine, purine orsynthetic analogs thereof, or a base linked to an amino acid, as in apeptide nucleic acid (PNA). A nucleotide is one monomer in apolynucleotide. A nucleotide sequence refers to the sequence of bases ina polynucleotide.

Oligonucleotide: An oligonucleotide is a plurality of joined nucleotidesjoined by native phosphodiester bonds, between about 6 and about 300nucleotides in length. An oligonucleotide analog refers to moieties thatfunction similarly to oligonucleotides but have non-naturally occurringportions. For example, oligonucleotide analogs can contain non-naturallyoccurring portions, such as altered sugar moieties or inter-sugarlinkages, such as a phosphorothioate oligodeoxynucleotide. Functionalanalogs of naturally occurring polynucleotides can bind to RNA or DNA,and include peptide nucleic acid (PNA) molecules.

Particular oligonucleotides and oligonucleotide analogs can includelinear sequences up to about 200 nucleotides in length, for example asequence (such as DNA or RNA) that is at least 6 bases, for example atleast 8, 10, 15, 20, 25, 30, 35, 40, 45, 50, 100 or even 200 bases long,or from about 6 to about 50 bases, for example about 10-25 bases, suchas 12, 15 or 20 bases.

Operably linked: A first nucleic acid sequence is operably linked with asecond nucleic acid sequence when the first nucleic acid sequence isplaced in a functional relationship with the second nucleic acidsequence. For instance, a promoter is operably linked to a codingsequence if the promoter affects the transcription or expression of thecoding sequence. Generally, operably linked DNA sequences are contiguousand, where necessary to join two protein-coding regions, in the samereading frame.

Open reading frame: A series of nucleotide triplets (codons) coding foramino acids without any internal termination codons. These sequences areusually translatable into a peptide.

Pharmaceutical agent: A chemical compound or composition capable ofinducing a desired therapeutic or prophylactic effect when properlyadministered to a subject or a cell. “Incubating” includes a sufficientamount of time for a drug to interact with a cell. “Contacting” includesincubating a drug in solid or in liquid form with a cell. An “anti-viralagent” or “anti-viral drug” is an agent that specifically inhibits avirus from replicating or infecting cells. Similarly, an“anti-retroviral agent” is an agent that specifically inhibits aretrovirus from replicating or infecting cells.

A “therapeutically effective amount” is a quantity of a chemicalcomposition or an anti-viral agent sufficient to achieve a desiredeffect in a subject being treated. For instance, this can be the amountnecessary to inhibit viral replication or to measurably alter outwardsymptoms of the viral infection, such as increase of T cell counts inthe case of an HIV infection. In general, this amount will be sufficientto measurably inhibit virus (e.g., HIV) replication or infectivity. Whenadministered to a subject, a dosage will generally be used that willachieve target tissue concentrations (for example, in lymphocytes) thathas been shown to achieve in vitro inhibition of viral replication.

Probes and primers: Nucleic acid probes and primers can be readilyprepared based on a nucleic acid sequence. A probe comprises an isolatednucleic acid attached to a detectable label or reporter molecule.Typical labels include radioactive isotopes, enzyme substrates,co-factors, ligands, chemiluminescent or fluorescent agents, haptens,and enzymes. Methods for labeling and guidance in the choice of labelsappropriate for various purposes are discussed, e.g., in Sambrook et al.(In Molecular Cloning: A Laboratory manual, CSHL, New York, 1989) andAusubel et al. (In Current Protocols in Molecular Biology, Greene Publ.Assoc. and Wiley-Intersciences, 1992).

Primers are short nucleic acid molecules, preferably DNAoligonucleotides 10 nucleotides or more in length. More preferably,longer DNA oligonucleotides can be about 15, 17, 20, or 23 nucleotidesor more in length. Primers can be annealed to a complementary target DNAstrand by nucleic acid hybridization to form a hybrid between the primerand the target DNA strand, and then the primer extended along the targetDNA strand by a DNA polymerase enzyme. Primer pairs can be used foramplification of a nucleic acid sequence, e.g., by the polymerase chainreaction (PCR) or other nucleic-acid amplification methods known in theart.

Methods for preparing and using probes and primers are described, forexample, in Sambrook et al. (In Molecular Cloning: A Laboratory Manual,CSHL, New York, 1989), Ausubel et al. (In Current Protocols in MolecularBiology, Greene Publ. Assoc. and Wiley-Intersciences, 1998), and Inniset al. (PCR Protocols, A Guide to Methods and Applications, AcademicPress, Inc., San Diego, Calif., 1990). PCR primer pairs can be derivedfrom a known sequence, for example, by using computer programs intendedfor that purpose such as Primer (Version 0.5, © 1991, WhiteheadInstitute for Biomedical Research, Cambridge, Mass.). One of ordinaryskill in the art will appreciate that the specificity of a particularprobe or primer increases with its length. Thus, for example, a primercomprising 30 consecutive nucleotides of the CD4 encoding nucleotidewill anneal to a target sequence, such as another nucleic acid encodingCD4, with a higher specificity than a corresponding primer of only 15nucleotides. Thus, in order to obtain greater specificity, probes andprimers can be selected that comprise at least 17, 20, 23, 25, 30, 35,40, 45, 50 or more consecutive nucleotides of cardiac nucleotidesequence of interest.

Protein: A biological molecule expressed by a gene and comprised ofamino acids.

Purified: The term “purified” does not require absolute purity; rather,it is intended as a relative term. Thus, for example, a purified proteinpreparation is one in which the protein referred to is more pure thanthe protein in its natural environment within a cell.

Recombinant: A recombinant nucleic acid is one that has a sequence thatis not naturally occurring or has a sequence that is made by anartificial combination of two otherwise separated segments of sequence.This artificial combination can be accomplished by chemical synthesisor, more commonly, by the artificial manipulation of isolated segmentsof nucleic acids, e.g., by genetic engineering techniques.

Sequence identity: The similarity between two nucleic acid sequences, ortwo amino acid sequences, is expressed in terms of the similaritybetween the sequences, otherwise referred to as sequence identity.Sequence identity is frequently measured in terms of percentage identity(or similarity or homology); the higher the percentage, the more similarthe two sequences are. Homologs or orthologs of the human CD4 protein,and the corresponding cDNA sequence, will possess a relatively highdegree of sequence identity when aligned using standard methods. Thishomology will be more significant when the orthologous proteins or cDNAsare derived from species which are more closely related (e.g., human andchimpanzee sequences), compared to species more distantly related (e.g.,human and murine sequences).

Methods of alignment of sequences for comparison are well known in theart. Various programs and alignment algorithms are described in: Smith &Waterman, Adv. Appl. Math. 2:482,1981; Needleman & Wunsch, J. Mol. Biol.48:443, 1970; Pearson & Lipman, Proc. Natl. Acad. Sci. USA 85:2444,1988; Higgins & Sharp, Gene, 73:237-244, 1988); Higgins & Sharp, CABIOS5:151-153, 1989; Corpet et al., Nuc. Acids Res. 16:10881-90, 1988; Huanget al., Computer Appls. in the Biosciences 8:155-65, 1992; and Pearsonet al., Meth. Mol. Bio. 24:307-31, 1994. Altschul et al., J. Mol. Biol.215:403-410, 1990, presents a detailed consideration of sequencealignment methods and homology calculations.

The NCBI Basic Local Alignment Search Tool (BLAST) (Altschul et al., J.Mol. Biol. 215:403-410, 1990) is available from several sources,including the National Center for Biotechnology Information (NCBI,Bethesda, Md.) and on the Internet, for use in connection with thesequence analysis programs blastp, blastn, blastx, tblastn and tblastx.It can be accessed at the NCBI website, together with a description ofhow to determine sequence identity using this program.

Homologs of the disclosed human CD4 protein typically possess at least60% sequence identity counted over full-length alignment with the aminoacid sequence of human CD4 using the NCBI Blast 2.0, gapped blastp setto default parameters. For comparisons of amino acid sequences ofgreater than about 30 amino acids, the Blast 2 sequences function isemployed using the default BLOSUM62 matrix set to default parameters,(gap existence cost of 11, and a per residue gap cost of 1). Whenaligning short peptides (fewer than around 30 amino acids), thealignment should be performed using the Blast 2 sequences function,employing the PAM30 matrix set to default parameters (open gap 9,extension gap 1 penalties). Proteins with even greater similarity to thereference sequence will show increasing percentage identities whenassessed by this method, such as at least 70%, at least 75%, at least80%, at least 90%, at least 95%, at least 98%, or at least 99% sequenceidentity. When less than the entire sequence is being compared forsequence identity, homologs will typically possess at least 75% sequenceidentity over short windows of 10-20 amino acids, and can possesssequence identities of at least 85% or at least 90% or 95% depending ontheir similarity to the reference sequence. Methods for determiningsequence identity over such short windows are described in the NCBIwebsite. These sequence identity ranges are provided for guidance only;it is entirely possible that strongly significant homologs could beobtained that fall outside of the ranges provided.

An alternative indication that two nucleic acid molecules are closelyrelated is that the two molecules hybridize to each other understringent conditions. Stringent conditions are sequence-dependent andare different under different environmental parameters. Generally,stringent conditions are selected to be about 5° C. to 20° C. lower thanthe thermal melting point (Tm) for the specific sequence at a definedionic strength and pH. The T_(m) is the temperature (under defined ionicstrength and pH) at which 50% of the target sequence remains hybridizedto a perfectly matched probe or complementary strand. Conditions fornucleic acid hybridization and calculation of stringencies can be foundin Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual, CSHL,New York and Tijssen (1993) Laboratory Techniques in Biochemistry andMolecular Biology—Hybridization with Nucleic Acid Probes Part I, Chapter2, Elsevier, N.Y. Nucleic acid molecules that hybridize under stringentconditions to a given CD4 sequence will typically under wash conditionsof 2×SSC at 50° C.

Nucleic acid sequences that do not show a high degree of identity cannevertheless encode similar amino acid sequences, due to the degeneracyof the genetic code. It is understood that changes in nucleic acidsequence can be made using this degeneracy to produce multiple nucleicacid molecules that all encode substantially the same protein.

T Cell: A white blood cell critical to the immune response. T cellsinclude, but are not limited to, CD4⁺ T cells and CD8⁺ T cells. A CD4⁺ Tlymphocyte is an immune cell that carries a marker on its surface knownas “cluster of differentiation 4” (CD4). These cells, also known ashelper T cells, help orchestrate the immune response, including antibodyresponses as well as killer T cell responses. CD8⁺ T cells carry the“cluster of differentiation 8” (CD8) marker. In one embodiment, a CD8 Tcells is a cytotoxic T lymphocytes. In another embodiment, a CD8 cell isa suppressor T cell.

Treatment: Refers to both prophylactic inhibition of initial infection,and therapeutic interventions to alter the natural course of anuntreated disease process, such as infection with a virus (e.g., HIVinfection).

Transformed: A transformed cell is a cell into which has been introduceda nucleic acid molecule by molecular biology techniques. As used herein,the term transformation encompasses all techniques by which a nucleicacid molecule might be introduced into such a cell, includingtransfection with viral vectors, transformation with plasmid vectors,and introduction of DNA by electroporation, lipofection, and particlegun acceleration.

Vector: A nucleic acid molecule as introduced into a host cell, therebyproducing a transformed host cell. Recombinant DNA vectors are vectorshaving recombinant DNA. A vector can include nucleic acid sequences thatpermit it to replicate in a host cell, such as an origin of replication.A vector can also include one or more selectable marker genes and othergenetic elements known in the art. Viral vectors are recombinant DNAvectors having at least some nucleic acid sequences derived from one ormore viruses.

Virus: Microscopic infectious organism that reproduces inside livingcells. A virus consists essentially of a core of a single nucleic acidsurrounded by a protein coat, and has the ability to replicate onlyinside a living cell. “Viral replication” is the production ofadditional virus by the occurrence of at least one viral life cycle. Avirus may subvert the host cells' normal functions, causing the cell tobehave in a manner determined by the virus. For example, a viralinfection may result in a cell producing a cytokine, or responding to acytokine, when the uninfected cell does not normally do so.

“Retroviruses” are RNA viruses wherein the viral genome is RNA. When ahost cell is infected with a retrovirus, the genomic RNA is reversetranscribed into a DNA intermediate which is integrated very efficientlyinto the chromosomal DNA of infected cells. The integrated DNAintermediate is referred to as a provirus. The term “lentivirus” is usedin its conventional sense to describe a genus of viruses containingreverse transcriptase. The lentiviruses include the “immunodeficiencyviruses” which include human immunodeficiency virus (HIV) type 1 andtype 2 (HIV-1 and HIV-2), simian immunodeficiency virus (SIV), andfeline immunodeficiency virus (FIV).

HIV is a retrovirus that causes immunosuppression in humans (HIVdisease), and leads to a disease complex known as the acquiredimmunodeficiency syndrome (AIDS). “HIV disease” refers to awell-recognized constellation of signs and symptoms (including thedevelopment of opportunistic infections) in persons who are infected byan HIV virus, as determined by antibody or western blot studies.Laboratory findings associated with this disease are a progressivedecline in T-helper cells.

The treatment of HIV disease has been significantly advanced by therecognition that combining different drugs with specific activitiesagainst different biochemical functions of the virus can help reduce therapid development of drug resistant viruses that were seen in responseto single drug treatment. In addition, discontinuation of existingtherapies results in a rapid rebound of viral replication, indicatingthe lack of complete HIV eradication by the drugs. There is therefore acontinuing need for the development of new anti-retroviral drugs thatact specifically at different steps of the viral infection andreplication cycle.

Fusion Proteins Including CD4, an Immunoglobulin Constant Region, and anAlpha Tailpiece

Disclosed herein are recombinant polypeptides comprising a CD4polypeptide ligated at its C-terminus with a portion of animmunoglobulin comprising a hinge region and a constant domain of amammalian immunoglobulin heavy chain. The portion of the immunoglobulinis ligated at its C-terminus with a polypeptide comprising a tailpiecefrom the C terminus of the heavy chain of an IgA antibody (αtp) or atailpiece from a C terminus of the heavy chain of an IgM antibody.

The term ligated encompasses the use of a linker between one polypeptidecomponent and another. Thus, in a specific, non-limiting example, alinker is included between a constant domain of the mammalianimmunoglobulin heavy chain and the tailpiece, (e.g., αtp). In anotherspecific, non-limiting example, a linker is included between the CD4polypeptide and the immunoglobulin constant domain. A linker includesany short chain polypeptide chain of between one and 35 amino acids,including but not limited to, a glycine repeat. One specific,non-limiting example of a linker is between one and ten glycineresidues, such as about two to about five glycine resides, or aboutthree glycine residues. Another example of a linker is gly-pro-prolinker or multimers of gly-pro-pro. The CD4 polypeptide can include theD1 and D2 regions of CD4 (e.g., see U.S. Pat. No. 5,126,433). Theboundary domains for the CD4 regions are, respectively, about 100-109(D1), about 175-184 (D2), about 289-298 (D3), and about 360-369 (D4),based on the precursor CD4 amino acid in which the initiating met isfound at −25 (see U.S. Pat. No. 6,117,655). Soluble CD4 molecules of useare also described in U.S. Pat. No. 5,422,274. In one embodiment, theCD4 molecule is any CD4 polypeptide, or functional fragment thereof,that binds gp120. In one, specific, non-limiting example, the CD4polypeptide is the D1 and D2 domains of the human CD4. In anotherspecific, non-limiting example, the CD4 polypeptide is a variant orfunctional fragment of the D1 and D2 domains of human CD4, chimpanzeeCD4 or rhesus macaque CD4. In another embodiment, the CD4 polypeptideincludes one or more modifications of the D1 and/or D2 domains such thatthe affinity of the CD4 for gp120 is altered. In one specific,non-limiting example, the affinity of the CD4 polypeptide for gp120 isincreased.

In one embodiment, the C-terminus of the CD4 polypeptide is ligated tothe N-terminus of a constant region of an immunoglobulin in place of thevariable region. As discussed above, the CD4 molecule can be directlyligated to the constant region (without the use of a linker), or alinker can be included between the CD4 and the constant region.

When the constant domain (Fc) is a heavy chain constant domain, all ofthe domains of constant region can be included. Typically, the fusionincludes at least the hinge region and one CH domain. In one embodiment,the fusion includes the hinge region and the CH2 and CH3 domain of theconstant region of an immunoglublin heavy chain.

The heavy chain constant region used in the construction of the CD4fusion protein can be from any antibody subclass, except IgA. Thus, theconstant region may be derived from an immunoglobulin of the IgG, IgD,IgE or IgM.subclass. When the Fc fragment is from an IgG antibody, anyof the human isotypes can be utilized (e.g., IgG₁, IgG₂, IgG₃, IgG₄). Inspecific, non-limiting example, the constant domain is from an IgG₂.Further, the parental IgG antibody or the isolated domain of theconstant region can be mutated to reduce binding to complement or Ig-Fcreceptors (e.g., see Duncan et al., Nature 332:563, 1988; Duncan andWinter, Nature 332:738, 1988; Alegre et al., J. Immunol. 148:3461, 1992;Tao et al., J. Exp. Med. 178:661, 1993; Xu et al., J. Bio. Chem.269:3469, 1994). In one embodiment, the constant region is from an IgM,and includes the hinge region, CH2 and CH3, but does not include thenaturally occurring 18 amino acids (μtail piece, but includes the αtp).In another embodiment, the Fc portion is from IgG, IgD, IgE, but notIgM, and the μtp is included.

In another embodiment, a chimeric constant domain is utilized thatincludes a domain of one isotype fused with another domain from adifferent isotype. One specific, non-liming example of a chimericconstant domain is the hinge of IgG₂ included in an IgG₁ constantdomain. In another embodiment, residues involved in the binding of anIgG₁ to a Fc region are replaced by residues from an IgG₂ that areinvolved in the binding to an Fc region. One of skill in the art canreadily identify residues involved in Fc binding (see Kabat et al.,supra, 1991; Shields et al., J. Biol Chem. 276(9): 6591-604, 2001). Inaddition, as the amino acids involved in contact between immunoglobulinsand Fc receptors (including, but not limited to, FcRN, FcγRII, andFcγRIII) can be identified, these specific residues can be substituted,using substitutions (such as in the formation of chimeras, ormutagenesis strategies). In one specific, non-limiting example, in theCH2CH3 region of an intact IgG₁ the following sequence

-   -   233-Glu Leu Leu Gly Gly Pro-238* (*using the numbering system        provided in Kabat et. al, supra, 1991)        is replaced by residues from the corresponding regions of an        immunoglobulin of a different class, such as, but not limited        to, IgG₂, IgA, IgM, or IgD. In one specific, non-limiting        example, corresponding residues from an intact IgG₂ are used,        such as, but not limited to:    -   233-Pro Val—Ala Gly Pro-238,        wherein “—” indicates the absence of a residue.

In another specific non-limiting example, one or more residues of IgG₁Fc binding domain are replaced from by a corresponding residue of animmunoglobulin from a different class. In yet another specific,non-limiting example, one or more residues of IgG₁ Fc binding domain arereplaced by any other amino acid, or are deleted. These residues,include, but are not limited to (Asn 297, Asp 265, Pro 329, Asp 270, Ala327, Ser 239, Lys 338) any other amino acid molecule (see Kabat, et al,supra, 1991 for a description of the numbering of amino acid moleculesin an immunoglobulin).

The CH2 or CH3 domain can also be modified by conventional techniques tocontain a restriction enzyme site for convenient cloning. In oneembodiment, the modified CD4 includes the D1 and D2 domains of CD4, thehinge, CH2 and CH3 domains of an IgG₂, wherein the CH3 domain ismodified to contain a restriction enzyme site for convenient cloning ofthe tailpiece of the heavy chain of an IgA antibody.

The tailpiece the heavy chain of an IgA antibody is a peptide located atthe C terminus of the naturally-occurring antibody. In one embodiment,this peptide is about eighteen residues in length. One peptide of use isPTHVNVSVVMAEDGTCY (SEQ ID NO:1). This peptide may be modified to removethe glycosylation site by changing one or more of the amino acid atresidues 5-7 (NVS, see above). this peptide is 18 amino acids in lengthand is derived from a human IgA molecule. In one embodiment, an alphatailpiece is PTHVNVSVVMAEVDGTCY (SEQ ID NO:1). However, if desired thepeptide may be modified to remove the glycosylation site by changing 1or 2 amino acids at residues 5-7 (NVS). For example, the asparagine (N)at position 5 can be changed to a glutamine (Q). Alternatively, theserine (S) at position 7 can be changed to an alanine (A). Additionally,a few of the amino acids residues of the IgA constant region may also beincluded, such as about four amino acids of the IgA constant region.

As discussed above, the fusion protein may include a linker sequencelocated between the constant domain of the immunoglobulin and the αtp.

Nucleic Acid Sequences and their Expression

The polypeptides disclosed herein be made using techniques known to oneof skill in the art (e.g., see WO 97/47732, herein incorporated byreference in its entirety). Briefly, each chain of the polypeptide isconstructed or selected. In one embodiment, the polypeptides disclosedherein are produced using recombinant technology. In one embodiment,nucleic acid sequences encoding the polypeptide of interest are producedand are expressed in vitro in a host cell. Variants of the nucleic acidsinclude variations due the degeneracy of the genetic code. Further, thepolynucleotide sequences may be modified by adding tags that can bereadily quantified, where desirable. Probes or primers can be used toassay the presence of the nucleic acid sequences in the host cell, orselectable markers can be included to facilitate detection of thenucleic acid sequences in a host cell.

The nucleic acid sequence encoding the fusion protein can also beinserted into other cloning vehicles, such as other plasmids,bacteriophages, cosmids, animal viruses and yeast artificial chromosomes(YACs) (Burke et al., Science 236:806-812, 1987). A suitable expressionsystem can be chosen to express the nucleic acid sequences in aeurkaryotic system. Numerous types of appropriate expression vectors andhost cell systems are known in the art, including, but not limited tosystems for expression in mammalian and yeast cells. Suitable host cellsor cell lines for transfection include human 293 cells, chines hamsterovary cells (CHO), murine L cells, monkey cells (e.g., COS-1 or COS-7),or murine 3T3 cells (e.g., see U.S. Pat. No. 4,419,449, or in Gethingand Sambrook, Nature 293:620, 1981, or in Kaufman et al., Mol. Cell.Biol. 5:1750, 1975).

For expression in mammalian cells, the nucleic acid sequence encodingthe CD4 fusion protein can be ligated to heterologous promoters, such asthe simian virus (SV) 40 promoter in the pSV2 vector (Mulligan and Berg,Proc. Natl. Acad. Sci. USA 78:2072-2076, 1981), and introduced intocells, such as monkey COS-1 cells (Gluzman, Cell 23:175-182, 1981), toachieve transient or long-term expression. The stable integration of thechimeric gene construct can be maintained in mammalian cells bybiochemical selection, such as neomycin (Southern and Berg, J. Mol.Appl. Genet. 1:327-341, 1982) and mycophenolic acid (Mulligan and Berg,Proc. Natl. Acad Sci. USA 78:2072-2076, 1981).

DNA sequences can be manipulated with standard procedures such asrestriction enzyme digestion, fill-in with DNA polymerase, deletion byexonuclease, extension by terminal deoxynucleotide transferase, ligationof synthetic or cloned DNA sequences, site-directed sequence-alterationvia single-stranded bacteriophage intermediate or with the use ofspecific oligonucleotides in combination with PCR.

The nucleic acid sequence can be introduced into eukaryotic expressionvectors by conventional techniques. These vectors are designed to permitthe transcription of the nucleic acid in eukaryotic cells by providingregulatory sequences that initiate and enhance the transcription of thecDNA and ensure its proper splicing and polyadenylation. Vectorscontaining the promoter and enhancer regions of the SV40 or longterminal repeat (LTR) of the Rous Sarcoma virus and polyadenylation andsplicing signal from SV40 are readily available (Mulligan et al., Proc.Natl. Acad. Sci. USA 78:1078-2076, 1981; Gorman et al., Proc. Natl.Acad. Sci USA 78:6777-6781, 1982). The level of expression of the cDNAcan be manipulated with this type of vector, either by using promotersthat have different activities (for example, the baculovirus pAC373 canexpress cDNAs at high levels in S.frugiperda cells (Summers and Smith, AManual of Methods for Baculovirus Vectors and Insect Cell CultureProcedures, Texas Agricultural Experiment Station Bulletin No. 1555,1987; Ausubel et al., Chapter 16 in Short Protocols in MolecularBiology, 1999) or by using vectors that contain promoters amenable tomodulation, for example, the glucocorticoid-responsive promoter from themouse mammary tumor virus (Lee et al., Nature 294:228, 1982).

In addition, some vectors contain selectable markers such as the gpt(Mulligan and Berg, Proc. Natl. Acad. Sci. USA 78:2072-2076, 1981) orneo (Southern and Berg, J. Mol. Appl. Genet. 1:327-341, 1982) bacterialgenes. These selectable markers permit selection of transfected cellsthat exhibit stable, long-term expression of the vectors (and thereforethe cDNA). The vectors can be maintained in the cells as episomal,freely replicating entities by using regulatory elements of viruses suchas papilloma (Sarver et al., Mol. Cell Biol. 1:486, 1981) orEpstein-Barr (Sugden et al., Mol. Cell Biol. 5:410, 1985).Alternatively, one can also produce cell lines that have integrated thevector into genomic DNA. Both of these types of cell lines produce theCD4 fusion protein on a continuous basis. One can also produce celllines that have amplified the number of copies of the vector (andtherefore of the cDNA as well) to create cell lines that can producehigh levels of the gene product (Alt et al., J. Biol. Chem. 253:1357,1978).

The transfer of DNA into eukaryotic, in particular human or othermammalian cells, is now a conventional technique. The vectors areintroduced into the recipient cells as pure DNA (transfection) by, forexample, precipitation with calcium phosphate (Graham and vander Eb,Virology 52:466, 1973) or strontium phosphate (Brash et al., Mol. CellBiol. 7:2013, 1987), electroporation (Neumann et al., EMBO J 1:841,1982), lipofection (Felgner et al., Proc. Natl. Acad. Sci. USA 84:7413,1987), DEAE dextran (McCuthan et al., J. Natl. Cancer Inst. 41:351,1968), microinjection (Mueller et al., Cell 15:579, 1978), protoplastfusion (Schafner, Proc. Natl. Acad. Sci. USA 77:2163-2167, 1980), orpellet guns (Klein et al., Nature 327:70, 1987). Alternatively, thecDNA, or fragments thereof, can be introduced by infection with virusvectors. Systems are developed that use, for example, retroviruses(Bernstein et al., Gen. Engr'g 7:235, 1985), adenoviruses (Ahmad et al.,J. Virol. 57:267, 1986), or Herpes virus (Spaete et al., Cell 30:295,1982). Sequences encoding a CD4 fusion protein can also be delivered totarget cells in vitro via non-infectious systems, for instanceliposomes. Thus disclosed herein are recombinant vectors that includenucleic acid encoding the CD4 fusion proteins, and host cell transfectedwith the vectors.

In another embodiment, transgenic animals are used to produce the CD4fusion proteins disclosed herein. One of skill in the art can readilyproduce transgenic mammals, including, but not limited to transgenicnon-human primates, transgenic sheep, transgenic cows, and transgenicmice containing a nucleic acid encoding the CD4 fusion proteins.Similarly transgenic plants can readily be produced that include nucleicacid sequences encoding the disclosed CD4 fusion proteins.

Pharmaceutical Compositions

The present invention includes a treatment for an infection withimmunodeficiency virus, in a subject such as an animal, for example amonkey or a human. Treatment includes both inhibition of initialinfection, and therapeutic interventions to alter the natural course ofan untreated HIV infection. The method includes administering the CD4fusion protein, or a combination of the CD4 fusion protein, andoptionally one or more other pharmaceutical agents, to the subject in apharmaceutically compatible carrier and in an amount effective toinhibit the development or progression of viral disease. In oneembodiment, the pharmaceutical agent is an anti-viral agent. In aspecific, non-limiting example, the anti-viral agent is ananti-retroviral agent, and the virus is HIV. In other, specific,non-limiting examples, the anti-viral agent is an anti-retroviral agent,and the virus is SIV or FIV. Although the treatment can be usedprophylactically in any patient in a demographic group at significantrisk for such diseases, subjects can also be selected using morespecific criteria, such as a definitive diagnosis of the condition.

The vehicle in which the drug is delivered can include pharmaceuticallyacceptable compositions of the drugs, using methods well known to thosewith skill in the art. Any of the common carriers, such as sterilesaline or glucose solution, can be utilized with the drugs provided bythe invention. Routes of administration include but are not limited tooral and parenteral routes, such as intravenous (iv), intraperitoneal(ip), subcutaneous, rectal, topical, ophthalmic, nasal, and transdermal.In one embodiment, a topical preparation is utilized. Suitableformulations for topical mircobicide preparations are known in the art.

The drugs may be administered intravenously in any conventional mediumfor intravenous injection, such as an aqueous saline medium, or in bloodplasma medium. The medium may also contain conventional pharmaceuticaladjunct materials such as, for example, pharmaceutically acceptablesalts to adjust the osmotic pressure, lipid carriers such ascyclodextrins, proteins such as serum albumin, hydrophilic agents suchas methyl cellulose, detergents, buffers, preservatives and the like. Amore complete explanation of parenteral pharmaceutical carriers can befound in Remington: The Science and Practice of Pharmacy (19^(th)Edition, 1995) in chapter 95.

Embodiments of other pharmaceutical compositions can be prepared withconventional pharmaceutically acceptable carriers, adjuvants andcounterions as would be known to those of skill in the art. Thecompositions are preferably in the form of a unit dose in solid,semi-solid and liquid dosage forms such as tablets, pills, powders,liquid solutions or suspensions.

The compounds of the present invention are ideally administered as soonas possible after potential or actual exposure to the virus.Alternatively the agent may be administered, for example, following anoccupational injury, such as a needle stick injury or involuntaryexposure to HIV infected blood. In another example, the agent can beadministered following high risk sexual activity. Alternatively, onceHIV infection has been confirmed by clinical observation or laboratorytests, a therapeutically effective amount of the CD4 fusion protein isadministered. In one embodiment, the dose can be given by frequent bolusadministration.

Therapeutically effective doses of the compounds of the presentinvention can be determined by one of skill in the art, with a goal ofachieving tissue concentrations that are at least as high as the IC₅₀ ofeach fusion protein. Low toxicity of the compound makes it possible toadminister high doses. An example of a dosage range is 0.01 to 100 mg/kgbody weight subcutaneously in single or divided doses. Another exampleof a dosage range is 0.1 to 100 mg/kg body weight subcutaneously insingle or divided doses. For oral administration, the compositions are,for example, provided in the form of a tablet containing 1.0 to 1000 mgof the active ingredient, particularly 1, 5, 10, 15, 20, 25, 50, 100,200, 400, 500, 600, and 1000 mg of the active ingredient for thesymptomatic adjustment of the dosage to the subject being treated.

The specific dose level and frequency of dosage for any particularsubject may be varied and will depend upon a variety of factors,including the activity of the specific compound, the metabolic stabilityand length of action of that compound, the age, body weight, generalhealth, sex, diet, mode and time of administration, rate of excretion,drug combination, and severity of the condition of the host undergoingtherapy.

Nucleotide based pharmaceuticals may be only inefficiently deliveredthrough ingestion. However, pill-based forms of pharmaceuticalnucleotides may be administered subcutaneously, particularly ifformulated in a slow-release composition. Slow-release formulations maybe produced by combining the target protein with a biocompatible matrix,such as cholesterol. Another possible method of administering thepharmaceuticals is through the use of mini osmotic pumps. As statedabove a biocompatible carrier would also be used in conjunction withthis method of delivery.

CD4 fusion proteins can be also be delivered to cells in the form of anucleic acid that encodes the CD4 fusion protein, and is subsequentlytranscribed by the host cell. When using this method to deliver a CD4fusion protein, a vector can be designed that contains a sequenceencoding the CD4 fusion protein. The vector can also include a promoterto drive the expression of the CD4 fusion protein. In one embodiment,the vector is a viral vector. The viral vector including nucleic acidencoding the CD4 fusion protein can be delivered as a virion or inconjunction with a liposome. Several techniques for deliveringtherapeutic nucleic acid sequences are well know in the art for example,Blau and Springer, New Engl. J. Med. 333:1204-1207, 1995, and Hanania etal., Amer. J. Med. 99:537-552, 1995.

A nucleic acid molecule can also be delivered directly to the cell vialiposome mediated delivery. The liposome fuses with or are enveloped bythe cells. Thus, a nucleic acid molecule encoding the CD4 fusion proteinis delivered intracellularly. Liposomes may be prepared with purifiedproteins or peptides that mediate fusion of membranes. Furthermore, theliposome may contain targeting molecules such as antibodies that allowthe liposome to selectively bind to specific cells within the body.Potential lipids that can be used in the formation of liposomes includeneutral lipids, such as cholesterol, phosphatidyl serine, phosphatidylglycerol, and the like. For preparing the liposomes, the proceduredescribed by Kato et al., J. Biol. Chem. 266:3361, 1991, may be used.

The pharmaceutical compositions of the present invention that includenucleic acids molecules may be administered by any means that achievetheir intended purpose (see above). For therapeutic use, an infectedcell is exposed to the polynucleotide in an effective concentration oreffective amount. Exposing a cell includes administering the molecule toany subject, such as a mammal (e.g., a human).

The pharmaceutical compositions can be used in the treatment of avariety of diseases caused by infection with viruses. Examples of suchdiseases include, but are not limited to, HIV-1 and HIV-2 infections.Other examples of such diseases include, but are not limited to SIV andFIV.

Combination Therapy

The present methods also include combinations of the CD4 fusion proteinsdisclosed herein with one or more antiviral drugs useful in thetreatment of viral disease. For example, the CD4 fusion proteinsdisclosed herein may be administered, whether before or after exposureto the virus, in combination with effective doses of other anti-virals,immunomodulators, anti-infectives, or vaccines. The term“administration” refers to both concurrent and sequential administrationof the active agents.

In one embodiment, a combination of CD4 fusion protein with one or moreagents useful in the treatment of a viral disease is provided. In onespecific, non-limiting example, the viral disease is a retroviraldisease, such as an HIV-1-induced, an HIV-2-induced, a SIV-induced, or aFIV induced disease.

Example of antivirals that can be used in the method of the inventionare: AL-721 (from Ethigen of Los Angeles, Calif.), recombinant humaninterferon beta (from Triton Biosciences of Alameda, Calif.), Acemannan(from Carrington Labs of Irving, Tex.), gangiclovir (from Syntex of PaloAlto, Calif.), didehydrodeoxythymidine or d4T (fromBristol-Myers-Squibb), EL10 (from Elan Corp. of Gainesville, Ga.),dideoxycytidine or ddC (from Hoffman-LaRoche), Novapren (from NovaferonLabs, Inc. of Akron, Ohio), zidovudine or AZT (from Burroughs Wellcome),ribavirin (from Viratek of Costa Mesa, Calif.), alpha interferon andacyclovir (from Burroughs Wellcome), Indinavir (from Merck & Co.), 3TC(from Glaxo Wellcome), Ritonavir (from Abbott), Saquinavir (fromHoffmann-LaRoche), and others.

Examples of immuno-modulators are AS-101 (Wyeth-Ayerst Labs.),bropirimine (Upjohn), gamma interferon (Genentech), GM-CSF (GeneticsInstitute), IL-2 (Cetus or Hoffman-LaRoche), human immune globulin(Cutter Biological), IMREG (from Imreg of New Orleans, La.), SK&F106528,TNF (Genentech), and soluble TNF receptors (Immunex).

Examples of some anti-infectives used include clindamycin withprimaquine (from Upjohn, for the treatment of pneumocystis pneumonia),fluconazlone (from Pfizer for the treatment of cryptococcal meningitisor candidiasis), nystatin, pentamidine, trimethaprim-sulfamethoxazole,and many others.

“Highly active anti-retroviral therapy” or “HAART” refers to acombination of drugs which, when administered in combination, inhibits aretrovirus from replicating or infecting cells better than any of thedrugs individually. In one embodiment, the retrovirus is a humanimmunodeficiency virus. In one embodiment, the highly activeanti-retroviral therapy includes the administration of3′axido-3-deoxy-thymidine (AZT) in combination with other agents.Examples of agents that can be used in combination in HAART for a humanimmunodeficiency virus are nucleoside analog reverse transcriptaseinhibitor drugs (NA), non-nucleoside analog reverse transcriptaseinhibitor drugs (NNRTI), and protease inhibitor drugs (PI). Onespecific, non-limiting example of HAART used to suppress an HIVinfection is a combination of indinavir and efavirenz, an experimentalnon-nucleoside reverse transcriptase inhibitor (NNRTI).

In one embodiment, HAART is a combination of three drugs used for thetreatment of an HIV infection, such as the drugs shown in Table 2 below.Examples of three drug HAART for the treatment of an HIV infectioninclude 1 protease inhibitor from column A plus 2 nucleoside analogsfrom column B in Table 2. In addition, ritonavir and saquinavir can beused in combination with 1 or 2 nucleoside analogs.

TABLE 2 Column A Column B indinavir (Crixivan) AZT/ddI nelfinavir(Viracept) d4T/ddI ritonavir (Norvir) AZT/ddC saquinavir (Fortovase)AZT/3TC ritonavir/saquinavir d4T/3TC

In addition, other 3- and 4-drug combinations can reduce HIV to very lowlevels for sustained periods. The combination therapies are not limitedto the above examples, but include any effective combination of agentsfor the treatment of HIV disease (including treatment of AIDS).

Induction of an Immune Response and Vaccines

The CD4 fusion proteins disclosed herein can be complexed to an HIV-1envelope protein and used to inhibit or prevent of HIV infection orinhibit disease progression. The CD4 fusion proteins can be used toinduce an immune response, such as, but not limited to, an immuneresponse against an HIV infected cell. In one specific, non-limitingexample, the immune response is activation of natural killer cells.

In one embodiment, a D1D2-Igαtp (see below) is complexed to an envelopeprotein of HIV1 and administered to a subject. Methods for forming acomplex of CD4 with an envelope protein of HIV (e.g., gp120) are known(e.g., see WO18433A2). In one embodiment, a CD4 fusion polypeptide iscovalently linked to gp120. One specific, non-limiting example of amethod for producing covalent linkage is chemical cross-lining. Inanother embodiment, a CD4 fusion polypeptide is non-covalently linked togp120. A specific, non-limiting example of a method for producingnon-covalent linkage is incubating the CD4 fusion polypeptide with gp120for a sufficient amount of time for a complex to form.

The conjugate is included in a vaccine formulation in an amount per unitdose sufficient to evoke an active immune response to the humanimmunodeficiency virus in the subject to be treated. The immune responsecan be any level of protection from subsequent exposure to the viruswhich is of some benefit in a population of subjects, whether in theform of decreased mortality, decreased morbidity, improved T cellnumbers or function, or the reduction of any other detrimental effect ofthe disease (e.g., reduction in HIV protease activity, see U.S. Pat. No.5,171,662), regardless of whether the protection is partial or complete.

The vaccine can be administered to the subject by any suitable means.Examples are by oral administration, intramuscular injection,subcutaneous injection, intravenous injection, intraperitonealinjection, eye drop or by nasal spray. Vaccine formulations canoptionally contain one or more adjuvants. Any suitable adjuvant can beused, including chemical and polypeptide immunostimulants which enhancethe immune system's response to antigens. Specific, non-limitingexamples of adjuvants are aluminum hydroxide, aluminum phosphate, plantand animal oils. These adjuvants are administered with the vaccineconjugate in an amount sufficient to enhance the immune response of thesubject to the vaccine conjugate. In addition, the vaccine formulationscan optionally contain one or more stabilizers. Any suitable stabilizercan be used, including, but not limited to carbohydrates such assorbitol, manitol, starch, sucrose, dextrin, or glucose; proteins suchas albumin or casein; and buffers such as alkaline metal phosphate andthe like. Methods for the preparation of vaccines are known in the art(see, for example, U.S. Pat. No. 6,136,319 and U.S. Pat. No. 6,113,962).

The dose of the vaccine may vary according to factors such as thedisease state, age, sex, immune status, and weight of the individual,and the ability to elicit a desired response in the individual. Dosageregime may be adjusted to provide the optimum therapeutic response. Forexample, several divided doses may be administered daily or the dose maybe proportionally reduced as indicated by the therapeutic situation. Thedose of the vaccine may also be varied to provide an optimumpreventative dose response depending upon the circumstances (see WO9703A1). Thus, a method for preventing an HIV infection, byadministering a CD4 fusion polypeptide, is provided herein.

In another embodiment, a DNA vaccine is utilized (e.g., see Nat. Med.,5(5):526-34, 1999). Thus, a method is provided for treating a viralinfection, such as an HIV infection, by providing a therapeuticallyeffective amount of a nucleic acid encoding a CD4 fusion polypeptide,such as, but not limited to a D1D2-Igαtp. Delivery of the polynucleotideencoding the CD4 fusion polypeptide can be achieved using a recombinantexpression vector such as a chimeric virus or a colloidal dispersionsystem, or through the use of targeted liposomes.

Various viral vectors which can be utilized for therapy include, but arenot limited to, adenoviral, herpes viral, or retroviral vectors. In oneembodiment, a retroviral vector such as a derivative of a murine oravian retroviral vector is utilized. Examples of retroviral vectors inwhich a single foreign gene can be inserted include, but are not limitedto: Moloney murine leukemia virus (MoMuLV), Harvey murine sarcoma virus(HaMuSV), murine mammary tumor virus (MuMTV), and Rous Sarcoma Virus(RSV). In addition, when the subject is a human, a vector such as thegibbon ape leukemia virus (GaLV) is utilized. A number of additionalretroviral vectors can incorporate multiple genes. The vectors cantransfer or incorporate a gene for a selectable marker so thattransduced cells can be identified and generated. By inserting a nucleicacid encoding the CD4 fusion polypeptide into the viral vector, alongwith another gene which encodes the ligand for a receptor on a specifictarget cell, for example, the vector is rendered target specific.Retroviral vectors can be made target specific by attaching, forexample, a sugar, a glycolipid, or a protein. Targeting can also beaccomplished by using an antibody to target the retroviral vector. Thoseof skill in the art will know of, or can readily ascertain without undueexperimentation, specific polynucleotide sequences which can be insertedinto the retroviral genome or attached to a viral envelope to allowtarget specific delivery of the retroviral vector.

Since recombinant retroviruses are defective, they require assistance inorder to produce infectious vector particles. This assistance can,beprovided, for example, by using helper cell lines that contain plasmidsencoding all of the structural genes of the retrovirus under the controlof regulatory sequences within the LTR. These plasmids are missing anucleotide sequence that enables the packaging mechanism to recognize anRNA transcript for encapsidation. Helper cell lines which have deletionsof the packaging signal include, but are not limited to Q2, PA317, andPA12, for example. These cell lines produce empty virions, since nogenome is packaged. If a retroviral vector is introduced into such cellsin which the packaging signal is intact, but the structural genes arereplaced by other genes of interest, the vector can be packaged andvector virion produced.

Alternatively, NIH 3T3 or other tissue culture cells can be directlytransfected with plasmids encoding the retroviral structural genes gag,pol and env, by conventional calcium phosphate transfection. These cellsare then transfected with the vector plasmid containing the genes ofinterest. The resulting cells release the retroviral vector into theculture medium.

Another targeted delivery system for therapeutic polynucleotides is acolloidal dispersion system. Colloidal dispersion systems includemacromolecule complexes, nanocapsules, microspheres, beads, andlipid-based systems including oil-in-water emulsions, micelles, mixedmicelles, and liposomes. Liposomes are artificial membrane vesicles thatare useful as delivery vehicles in vitro and in vivo. It has been shownthat large unilamellar vesicles (LUV), which range in size from 0.2-4.0μm, can encapsulate a substantial percentage of an aqueous buffercontaining large macromolecules. RNA, DNA and intact virions can beencapsulated within the aqueous interior and be delivered to cells in abiologically active form (Fraley et al., Trends Biochem. Sci. 6:77,1981). In order for a liposome to be an efficient gene transfer vehicle,the following characteristics should be present: (1) encapsulation ofthe genes of interest at high efficiency while not compromising theirbiological activity; (2) preferential and substantial binding to atarget cell in comparison to non-target cells; (3) delivery of theaqueous contents of the vesicle to the target cell cytoplasm at highefficiency; and (4) accurate and effective expression of geneticinformation (Mannino et al., Biotechniques 6:682, 1988; see also U.S.Pat. No. 6,270,795).

The composition of the liposome is usually a combination ofphospholipids, particularly high-phase-transition-temperaturephospholipids, usually in combination with steroids, such ascholesterol. Other phospholipids or other lipids may also be used. Thephysical characteristics of liposomes depend on pH, ionic strength, andthe presence of divalent cations. Examples of lipids useful in liposomeproduction include phosphatidyl compounds, such as phosphatidylglycerol,phosphatidylcholine, phosphatidylserine, phosphatidylethanolamine,sphingolipids, cerebrosides, and gangliosides. Particularly useful arediacylphosphatidyl-glycerols, where the lipid moiety contains from 14-18carbon atoms, particularly from 16-18 carbon atoms, and is saturated.Illustrative phospholipids include egg phosphatidylcholine,dipalmitoylphosphatidylcholine and distearoylphosphatidylcholine.

The targeting of liposomes can be classified based on anatomical andmechanistic factors. Anatomical classification is based on the level ofselectivity, for example, organ-specific, cell-specific, andorganelle-specific. Mechanistic targeting can be distinguished basedupon whether it is passive or active. Passive targeting utilizes thenatural tendency of liposomes to distribute to cells of thereticulo-endothelial system (RES) in organs which contain sinusoidalcapillaries. Active targeting, on the other hand, involves alteration ofthe liposome by coupling the liposome to a specific ligand such as amonoclonal antibody, sugar, glycolipid, or protein, or by changing thecomposition or size of the liposome in order to achieve targeting toorgans and cell types other than the naturally occurring sites oflocalization.

The surface of the targeted delivery system may be modified in a varietyof ways. In the case of a liposomal targeted delivery system, lipidgroups can be incorporated into the lipid bilayer of the liposome inorder to maintain the targeting ligand in stable association with theliposomal bilayer. Various linking groups can be used for joining thelipid chains to the targeting ligand.

In one embodiment, a mammalian subject, is administered atherapeutically effective dose of a pharmaceutical compositioncontaining nucleic acid encoding a CD4 polypeptide ligated at itsC-terminus with a portion of an immunoglobulin comprising a hinge regionand a constant domain of a mammalian immunoglobulin heavy chain, whereinthe portion of the immunoglobulin is fused at its C-terminus with apolypeptide comprising a tailpiece from the C terminus of the heavychain of an IgA antibody in a pharmaceutically acceptable carrier.

The CD4 fusion proteins disclosed herein can be used to generate animmune response against an HIV infected cells. In one specific,non-limiting example, the response is antibody dependent cell mediatedcytotoxicity. Thus, a method is disclosed herein for inducing antibodydependent cell-mediated cytotoxicity of a cell infected with alentivirus, such as an immunodeficiency virus (e.g. SIV, HIV-1, HIV-2 orFIV). The method includes contacting the cell with an effective amountof the CD4 fusion protein in the presence of an antigen presenting cell(macrophage, dendritic cell, neutrophil, or B-lymphocyte) or a naturalkiller cell, thereby stimulating the antigen presenting cell or thenatural killer cells, and inducing antibody dependent cell mediatedcytotoxicity of the cell infected with the immunodeficiency virus. Theimmunodeficiency virus can be a human immunodeficiency virus (e.g. HIV-1or HIV-2). The response (e.g. stimulation of the antigen presenting cellor natural killer cell, inducing antibody dependent cell mediatedcytotoxicity, or both) can be produced either in vitro, ex vivo, or invivo. In one specific, non-limiting example, the natural killer cell orthe antigen presenting cell (APC) isolated from a subject, and iscontacted with the CD4 fusion protein in vitro. The stimulated naturalkiller cell and/or APC is subsequently transferred into the subject ofinterest.

Thus, a method is disclosed herein for stimulating a natural killer cellresponse or the stimulation of an antigen presenting cell. The methodincludes contacting the natural killer cell with an effective amount ofa CD4 fusion protein, thereby stimulating the response of the naturalkiller cell. One of skill in the art can readily identify assays fornatural killer cell activation, such as, but not limited to, assays ofcalcium flux and cytoxicity assays. Examples of an assay for naturalkiller cell activation are provided in the Examples section (see below).

Screening Assays

The CD4 fusion proteins disclosed herein are of use for in vitro assaysfor measuring the binding of the fusion CD4 fusion protein to a selectedviral isolate and for identifying affinity of a particular gp120 forCD4. In one embodiment, a biosensor assay is utilized (e.g., see FIG. 6and the Examples). In another embodiment, an ELISA assay is utilized.

The CD4 fusion protein may be used in assays to screen for new compoundsthat inhibit HIV replication. In one specific, non-limiting example, aviral isolate or an isolated gp120 is contacted with the CD4 fusionprotein in the presence of an agent of interest. The ability of thevirus or the gp120 to bind to the CD4 fusion protein is then assessed.Agents of interest include, but are not limited to polypeptides,isolated biological material, chemical compounds, pharmaceutical, orpeptidomimetics.

In one embodiment, the CD4 fusion protein is labeled. In anotherembodiment, the CD4 fusion protein is immobilized on a solid substrate,and the gp120 is labeled. Suitable labels include, but are not limitedto, enzymatic, fluorescent, or radioactive labels. Alternatively eitherthe CD4 fusion polypeptide (e.g., D1D2-Igαtp) or the gp120 is covalentlylinked to a biosensor chip. Either gp120, or the CD4 fusion polypeptide(e.g., D1D2-Igαtp), respectively, is then passed over the chip. Thistype of assay can is readily adapted to high throughput screening foreither synthetic or natural compounds that interfere with theinteraction of gp120 and the CD4 fusion polypeptide.

Without further elaboration, it is believed that one skilled in the artcan, using this description, utilize the present invention to itsfullest extent. The following examples are illustrative only, and notlimiting of the remainder of the disclosure in any way whatsoever.

EXAMPLES Example 1 Materials and Methods

Virus entry. Virion entry into primary lymphocytes was measured using aquantitative real-time PCR assay based upon the generation of early LTRtranscripts, adapted from a method previously described (Chun et al.,Nature 387(6629):183, 1997). Briefly, freshly isolated peripheral bloodmononuclear cells (PBMCs) were activated (OKT3 (1 μg/ml)/IL2 (50 u/ml)for three days and then depleted of CD8+ T-cells by magnetic beadselection (Dynal, Lake Success N.Y.). 3×10⁶ cells were incubated in avolume of 100 μl with the addition of titered viral stocks (AdvancedBiotechnologies Columbia, Md.) at a multiplicity of infection (MOI) of0.1 for two hours at 37° C. Where specified, monomeric sCD4 andD1D2-Igαtp (see below) were preincubated with virus stocks for 10minutes at 37° C. prior to cell inoculation. Cells were washed with PBS,pelleted through a 100% fetal bovine serum (FBS) cushion (heatinactivated), and then resuspended in DMEM/FBS (heat inactivated) andincubated an additional 4 hrs. Cells were washed and then lysed in abuffer containing an anionic detergent (Gentra, Minneapolis, Minn.) andRNase-A. DNA was precipitated from lysates in isopropanol andresuspended in dH₂0. Quantitative real-time PCR was carried out usingthe following primers and probe: RU5 forward primer:5′-gctaactagggaacccactgctt-3′ (SEQ ID NO:2), RU5 reverse primer:5′-acaacagacgggcacacactact-3′ (SEQ ID NO:3), RU5 probe:5′-agcctcaataaagcttgccttgagtgcttc-3′ (SEQ ID NO:4). Copy numbers werestandardized against genomic DNA obtained from an ACH-2 cell linecarrying a single integrated HIV-1 genome in each diploid cell (Folks etal., Science 231(4738):600, 1986).

Expression and purification of D1D2-Igαtp. The two N-terminal domains ofCD4, termed D1 and D2 encode the gp120 binding epitope, and whenexpressed in the absence of the remaining domains of CD4, retain thecapacity to bind gp120. The coding sequences of D1 and D2 was fused tothat of Igαtp creating a recombinant protein termed D1D2-Igαtp Thecoding sequence of this protein is:

    ATGAACCGGGGAGTCCCTTTTAGGCACTTGCTTCTGGTGCTGCAA (SEQ ID NO:5)CTGGCGCTCCTCCCAGCAGCCACTCAGGGAAAGAAAGTGGTGCTGGGCAAAAAAGGGGATACAGTGGAACTGACCTGTACAGCTTCCCAGAAGAAGAGCATACAATTCCACTGGAAAAACTCCAACCAGATAAAGATTCTGGGAAATCAGGGCTCCTTCTTAACTAAAGGTCCATCCAAGCTGAATGATCGCGCTGACTCAAGAAGAAGCCTTTGGGACCAAGGAAACTTCCCCCTGATCATCAAGAATCTTAAGATAGAAGACTCAGATACTTACATCTGTGAAGTGGAGGACCAGAAGGAGGAGGTGCAATTGCTAGTGTTCGGATTGACTGCCAACTCTGACACCCACCTGCTTCAGGGGCAGAGCCTGACCCTGACCTTGGAGAGCCCCCCTGGTAGTAGCCCCTCAGTGCAATGTAGGAGTCCAAGGGGTAAAAACATACAGGGGGGGAAGACCCTCTCCGTGTCTCAGCTGGAGCTCCAGGATAGTGGCACCTGGACATGCACTGTCTTGCAGAACCAGAAGAAGGTGGAGTTCAAAATAGACATCGTGGTGCTAGCTTCGGCCGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGGGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCGCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTAAGCTTGTCTGCGGGTAAACCCACCCATGTCAATGTGTCTGTTGTCATGGCGGAGGTGGACGGCACCTGCTACTGAThis amino acid sequence corresponding to this coding sequence is:

MNRGVPFRHLLLVLQLALLPAATQGKKVVLGKKGDTVELTCTASQKKSIQFH (SEQ ID NO:6) WKNSNQIKILGNQGSFLTKGPSKLNDRADSRRSLWDQGNFPLIIKNLKIED SDTYICEVEDQKEEVQLLVFGLTANSDTHLLQGQSLTLTLESPPGSSPSVQ CRSPRGKNIQGGKTLSVSQLELQDSGTWTCTVLQNQKKVEFKIDIVVLASA DKTHTCPPCPAPELLGGPSVFLFPPKPDTLMISRTPEVTCVVVDVSHEDPE VKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKV SNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYP SDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSC SVMHEALHNHYTQKSLSLSAGKPTHVNVSVVMAEVDGTCYThe D1D2 domains of CD4 encoded in this construct include the followingsequence:

ATGAACCGGGGAGTCCCTTTTAGGCACTTGCTTCTGGTGCTGCAACTGGC (SEQ ID NO:7) GCTCCTCCCAGCAGCCACTCAGGGAAAGAAAGTGGTGCTGGGCAAAAAA GGGGATACAGTGGAACTGACCTGTACAGCTTCCCAGAAGAAGAGCATAC AATTCCACTGGAAAAACTCCAACCAGATAAAGATTCTGGGAAATCAGGG CTCCTTCTTAACTAAAGGTCCATCCAAGCTGAATGATCGCGCTGACTCA AGAAGAAGCCTTTGGGACCAAGGAAACTTCCCCCTGATCATCAAGAATC TTAAGATAGAAGACTCAGATACTTACATCTGTGAAGTGGAGGACCAGAA GGAGGAGGTGCAATTGCTAGTGTTCGGATTGACTGCCAACTCTGACACC CACCTGCTTCAGGGGCAGAGCCTGACCCTGACCTTGGAGAGCCCCCCTG GTAGTAGCCCCTCAGTGCAATGTAGGAGTCCAAGGGGTAAAAACATAGA GGGGGGGAAGACCCTCTCCGTGTCTCAGCTGGAGCTCCAGGATAGTGGC ACCTGGACATGCACTGTCTTGCAGAACCAGAAGAAGGTGGAGTTCAAAA TAGACATCGTGGTGCTAGCTTTCGGCCGThe coding sequence of the IgG1α tailpiece fusion is:

TCGGCCGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCT (SEQ ID NO:8) GGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTC ATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCC ACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGT GCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTAC CGGGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCA AGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGA GAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTAC ACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGA CCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGA GAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTG GACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGA GCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGC TCTGCACAACCACTACACGCAGAAGAGCCTAAGCTTGTCTGCGGGTAAA CCCACCCATGTCAATGTGTCTGTTGTCATGGCGGAGGTGGACGGCACCT  GCTACTGAThe coding sequence for this sequence is:

SADKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPE (SEQ ID NO:9) VKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSN KALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIA VEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSGSVMHEA LHNHYTQKSLSLSAGKPTHVNVSVVMAEVDGTCY*

The D1D2-Igαtp is predicted to be a hexamer of a dimer (12 bindingsites) of sCD4 that we compared to a monomeric sCD4. The D1D2-Igαtpexpression vector was designed using standard recombinant DNAmethodologies (Chaikin et al., AAAAI, San Francisco, Calif., 1997; J.Sambrook, E. F. F. et al., Cold Spring Harbor Laboratory Press,Plainview, N.Y., 1989). This vector contains a CMV promoter for highlevel expression of D1D2-Igαtp as well as a gene cassette containingdihydrofolate reductase (DHFR) for amplification in DHFR deficientChinese hamster ovary (CHO) cells (American type culture collectioncatalogue (ATCC) No. CRL9096). Purified plasmids were transfected intoDHFR deficient CHO cells by a modified Calcium phosphate transfectionprocedure (Invitrogen, Carlsbad, Calif.). Positive transfectants wereinitially selected by growth in alpha-MEM without nucleosidessupplemented with dialyzed fetal calf serum (Life Technologies,Baltimore, Md.). To increase expression, positive transfectants werepooled and cultured in the presence of increasing concentrations ofmethotrexate (Sigma, St. Louis Mo.) as previously described (Arthos etal., Cell 57, No. 3:469, 1989). Cell clones expressing high levels ofD1D2-Igαtp were identified by Western blot with a rabbit polyclonalantisera raised against sCD4. Clones were subsequently cultured inhollow fiber cartridges (FiberCell Systems, Frederick, Md.) using DMEMplus 4% heat inactivated FBS without methotrexate. Proteins wereharvested daily from the extra-capillary space, yielding greater than 5mg per harvest. D1D2-Igαtp protein was purified in two steps. Initially,supernatants from the extra capillary space of the hollow fibercartridge were passed over a Hi-trap protein A column(Amersham-Pharmacia Piscataway, N.J.). Bound protein was eluted in 0.1Msodium citrate pH 3.0 and rapidly neutralized with 2M tris-HCL pH 8.0Peak fractions were subsequently pooled, concentrated and passed overeither a Superdex Hi-load 26/60 or a Superdex 200 10/30 gel filtrationcolumn (Amersham-Pharmacia Piscataway, N.J.) in PBS, and the peakfraction was collected. With the exception of analyticalultracentrifugation and dynamic light scattering experiments, this wasthe fraction employed in all biological assays. Silver staining ofSDS-page gels indicated that the purity of protein obtained in thismanner was >98%. Protein preparations were determined to be endotoxinfree using the Chromogenic Limulus Amebocyte Lysate method (BioWhittaker, Walkersville, Md.)

Optical Biosensor Analysis. General Procedures: All binding assays wereperformed using a BIA3000 optical biosensor (Biacore, Inc., Uppsala,Sweden). Ligands were immobilized onto the surface of a CM5 sensor chipusing the standard amine coupling procedure described by Biacore, Inc.Briefly, the carboxyl groups on the sensor surface were activated byinjecting 35 μl of 0.2 M N-ethyl-N′-(3-diethylamino-propyl) carbodiimide(EDC), 0.05 M N-hydroxy-succinimide (NHS). The ligand, suspended in 10nM acetate buffer, pH 4.0-5.5 (depending on the ligand used) to 5 μg/mL,was passed over the activated surface until the desired surface densitywas reached. Unreacted carboxyl groups were capped by injecting 35 μl of1 M ethanolamine (pH 8.0). All samples were injected at a flow rate of 5microliters/min Bovine serum albumin (BSA) was immobilized on thesurface of one flow cell as a reference surface to control fornon-specific binding of analyte. The running buffer used was 10 mMHEPES, pH 7.4, 150 mM NaCl, 3 mM EDTA, 0.01% surfactant P-20, 0.5%soluble carboxymethyl dextran (Fluka BioChemika, Inc.). All bindingexperiments were performed in duplicate and at 25° C.

Interaction Analysis Between sCD4 or D1D2-Igαtp and HIV-1 gp120: sCD4 orHIV-1 gp120 were directly immobilized onto the surface of CM5 sensorchips as described above to surface densities of approximately 250 RUfor sCD4 and 500 RU for gp120. This was followed by injection ofincreasing concentrations of gp120 or sCD4, respectively, throughmultiple cycles, at a flow rate of 25 microliters/min. The surface wasregenerated after each cycle by injecting 25 μl of 5 mM NaOH, 1 M NaCl,followed by a second injection of 25 μl of 4.5 M MgCl₂ at a flow rate of100 microlitersL/min. Association and dissociation rate constants werecalculated using the BiaEvaluation 3.1 software (Biacore, Inc., Uppsala,Sweden).

Determination of D1D2-Igαtp: gp120 Binding Ratios: To determine theratio of gp120 monomers bound per D1D2-Igαtp construct, D1D2-Igαtp inrunning buffer was passed over a sensor surface to which protein A hadbeen previously immobilized (surface density approximately 1500 RU) at aflow rate of 5 microliters/min. The final surface density of D1D2-Igαtpwas approximately 250 RU, and was re-loaded to this density at thebeginning of each cycle. After loading of D1D2-Igαtp, the surface wasallowed to stabilize for 5 minutes at a flow rate of 5 microliters/min,at which time the specified concentrations of gp120 in running bufferwas passed over the surface for a total of 10 minutes. The surface wascompletely regenerated using three sequential 25 μl injections of 10 mMHCl at a flow rate of 100 microliters/min. Stoichiometries werecalculated from the experimentally derived amount of D1D2-Igαtp andgp120 bound per cycle (in RU) using the conversion factor 1 RU=1 pgprotein bound per square millimeter flow cell surface area, and themolecular weights of the proteins (D1D2-Igαtp=750,000 Da, gp120=120,000Da).

Virus coculture. Virus coculture was carried out as previously described(Chun et al., Nature 387:183, 1997). Briefly, peripheral bloodmononuclear cells (PBMCs) from HIV-1 infected donors were isolated byFicoll-Hypaque and enriched for CD4+ T-lymphocytes by negative selectionwith a cocktail of antibody conjugated magnetic beads (StemCellTechnologies, Vancouver BC). Cells were cultured in RPMI/10% FBS (heatinactivated) plus OKT3 (1 μg/ml) and IL2 (50 u/ml). In addition, 3 dayactivated CD8+ T-cell depleted PBMCs from uninfected donors were addedat a ratio of approximately 2:1 as necessary. Cultures treated withmonomeric sCD4 or D1D2-Igαtp were fed with media containing theseproteins such that the original concentration was maintained. Virusreplication was assessed by harvesting culture supernatants at regularintervals and measuring p24 antigen using an HIV-1 p24 Antigen CaptureKinetic ELISA (Coulter, Miami, Fla.).

Acute infection. Freshly isolated donor PBMCs were propagated in RPMIsupplemented with 10% FBS and stimulated with OKT3 (1 μg/ml) and IL2 (50u/ml). Prior to infection cells were screened by PCR for CCR5 wild-typehomozygosity. Three days after stimulation CD8+ cells were depleted bymagnetic bead separation (Dynal, Lake Success, N.Y.), and inoculatedwith virus as indicated at an MOI of 0.1. Primary isolates wereestablished from six day coculture of patient and normal donor CD8depleted PBMCs. Cells were exposed to virus for two hours at 37° C., andthen washed extensively in PBS. Cells were then plated at a density of2×10⁶ cells per ml in 24 well tissue culture plates. Immediately afterplating various inhibitors were added. Supernatants were collected everyother day and virus replication was measured by a kinetic p24 antigencapture ELISA (Coulter, Miami, Fla.) Inhibitor concentrations weremaintained in the culture supernatants throughout the culture period.

Analytical Ultracentrifugation and Dynamic Light Scattering.Sedimentation velocity experiments were conducted with the analyticalultracentrifuge Beckman Optima XL-I/A using interference optics, with400 micrograms of protein (1 μg/μl) dissolved in PBS at a rotor speed of30,000 rpm and a rotor temperature of 20° C. Data were analyzed bydirect boundary modeling with a continuous distribution of Lamm equationsolutions (Schuck, Biophys J 78, No.3:1606, 2000) and algebraic noisedecomposition (Schuck et al., Biophys J 76, No. 4:2288, 1999). Thedistribution of Lamm equation solutions c(s) were calculated withmaximum entropy regularization with P=0.68. For deconvolution of thediffusion, the best-fit average frictional ratio of 1.5 was used,resulting in rms deviations of the direct boundary fit of <0.004 fringesin all cases. Sedimentation equilibrium experiments were performed withthe absorbance optics at a wavelength of 280 nanometer (nm) and a rotortemperature of 4° C. Equilibrium was attained at rotor speeds of 3,000rpm, 5,000 rpm, and 7,500 rpm with best-fit distributions with asingle-species model for the determination of the weight-average molarmass (Svedberg, T.a.K.O.P., Oxford University Press, London U.K, 1940).

Using tabulated values of the partial specific volume of amino acids(Laue et al., The Royal Society of Chemistry, Cambridge U.K. (1992)) and0.62±0.02 ml/g for the average partial specific volume of thecarbohydrate component (Lewis et al., Methods Enzymol 321:136, 2000),and with an average glycosylation of 5 and 15 kDa at the twoglycosylation sites per chain, a molar mass of 140 kDa and a partialspecific volume of 0.699 ml/g was estimated for a monomeric unit (twochains). Dynamic light scattering experiments were conducted using aProtein Solutions DynaPro 99 instrument with a DynaPro-MSTC200microsampler (Protein Solutions, Charlottesville, Va.). 20 μl of samplewas inserted in the cuvette with the temperature control set to 20° C.The light scattering signal was collected at 90° C. at a wavelength of808.3 nm. Data analysis was performed with the instrument software, andexported for analysis with the maximum entropy method (Livesey et al.,J. Chem. Phys. 84:5102, 1986, in the software SEDFIT (Schuck, Biophys J78, No. 3:1606, 2000).

Example 2 Expression of a CD4 Fusion Protein

D1D2-Igαtp is expressed as a highly oligomerized protein. It was firstasked whether D1D2-Igαtp was expressed in a highly oligomerized form. Tothis end, was purified from culture supernatants by protein-A affinitychromatography, and analyzed by standard size exclusion chromatography.When passed over an analytic superdex-200 gel-filtration column a majorpeak appeared in that fraction corresponding to a molecular weightgreater than 650 kDa. A minor fraction, comprising less than 5% of totalprotein eluted in the 50-100 kDa range. Because the major fractionappeared close to the void volume of the column, it was not possible toaccurately estimate its molecular weight from these data. Thesefractions were then reduced and electrophoresed under denaturingconditions. Western blot analysis with a polyclonal antisera specificfor CD4 indicated that D1D2-Igαtp resided primarily in the peak fraction(FIG. 1). When this blot was re-probed with a goat anti-human Ig serasimilar results were observed. When the peak fractions were displayed ona Coomassie stained gel, one major band with an approximate molecularweight of 60 kDa was observed. This molecular weight is in closeagreement with the unit molecular weight of D1D2-Igαtp predicted byamino acid composition. Thus, D1D2-Igαtp is expressed as a highlyoligomerized protein presenting both CD4 and human immunoglobulin heavychain domains.

Example 3 Comparison of D1D2-Igαtp and Monomeric sCD4 in a QuantitativeHIV Entry Assay

To determine the efficiency with which D1D2-Igαtp inhibited HIV entry areal-time PCR based quantitative viral entry assay was established.Virion entry was detected by measuring the level of the initial reversetranscription products in the R and U5 regions of the HIV-1 LTR. Thetarget cells utilized in this assay were three day activated, CD8+T-cell depleted PBMCs. After optimization, the linear range of thisassay typically fell between 25 and 200,000 copies of reversetranscribed product. Two viruses, JR-FL and Bal, both of which utilizethe CCR5 coreceptor and were derived after minimal passage of primaryisolates, were then employed. To establish the conditions under whichsCD4 would enhance viral entry, viral inoculi were briefly pre-incubatedwith various concentrations of monomeric sCD4 and then carried out theentry assay.

Under these conditions sCD4 at a concentration of either 6.25 or 12.5 nMrepeatedly increased viral entry by 1.5 to 3 fold for both JR-FL (FIG.2) and Bal. At high concentrations (2400 nM) sCD4 reduced viral entry tolevels close to background. In contrast across this entire range ofconcentrations, D1D2-Igαtp reduced virus entry down to levels close tobackground (FIG. 2A). Thus, at the concentrations in which sCD4 providesoptimal enhancement of viral entry D1D2-Igαtp strongly inhibits viralentry. Because each D1D2-Igαtp molecule presents multiple gp120 bindingsites the possibility was considered that it might enhance entry at evenlower concentrations. D1D2-Igαtp was titered down to 50 picomolar (pM);however, enhanced viral entry (FIG. 2B) was not observed. Therefore itwas concluded that unlike sCD4, D1D2-Igαtp does not enhance viral entryat low concentrations.

Example 4 D1D2-Igαtp Versus Monomeric sCD4 Inhibition of Primary ViralIsolates from Patient PBMCs

The capacity of monomeric sCD4 and D1D2-Igαtp to inhibit the replicationof HIV-1 in cultures of PBMCs derived from HIV-1 infected patients wasassessed. CD4+ T cells were isolated from patients and placed intoculture along with activated PBMCs from uninfected donors. To thesecultures concentrations of monomeric sCD4 were added that enhanced entryof Bal and JR-FL in the viral-entry assay (FIG. 3). In two of the threecocultures the addition of sCD4 resulted in enhanced replication (FIGS.3A, 3B, and 3C), while in the third coculture sCD4 inhibited viralreplication to a limited degree (FIG. 3D.). The same donor CD4+ T-cellswere treated in parallel with D1D2-Igαtp. At the same concentrations ofmonomeric sCD4 that enhanced viral replication in two of three donorcells, D1D2-Igαtp strongly inhibited viral replication in all threedonor cells (FIG. 3). Thus, unlike monomeric sCD4, which enhances viralreplication at low concentrations, D1D2-Igαtp actively inhibits thereplication of primary isolates of HIV-1 in activated T-cells.

Example 5 D1D2-Igαtp Inhibits Monoclonal Antibody (mAb) MediatedEnhancement of HIV-1 Replication

Similar to sCD4, a number of mAbs specific for gp120 have been shown toenhance replication of HIV-1 at suboptimal concentrations (Sullivan etal., J Virol 69, No. 7:4413, 1995; Schutten et al., Scand J Immunol 41,No. 1:18, 1995; Sullivan et al., J. Virol 72, No. 6:4694, 1998). One ofthese mAbs, termed 17b, recognizes an epitope on gp120 that overlaps theCCR5 binding site (Kwong et al., Nature 393, No. 6686:648, 1998). Thisepitope is exposed subsequent to envelope-CD4 ligation. Consequently,17b reacts more efficiently with gp120 in the presence of sCD4. It wasassessed whether D1D2-Igαtp could prevent 17b-mediated enhancement ofviral replication. PBMCs were acutely infected with either Bal or aprimary isolate derived from a patient shortly after seroconversion.Parallel cultures were treated with 17b, sCD4, 17b plus sCD4, D1D2-Igαtpor 17b plus D1D2-Igαtp, and the extent of viral replication wasdetermined by measurement of p24 antigen in culture supernatants. Forboth Bal and the primary isolate, 17b alone enhanced viral replicationrelative to control cultures (FIG. 4). The combination of 17b plus sCD4also resulted in enhanced replication relative to control cultures. sCD4alone enhanced replication of Bal to a modest degree. Surprisingly, thecombination of sCD4 and 17b appeared to enhance Bal replication in anadditive manner (FIG. 4A), suggesting that higher concentrations of oneor both of these ligands would be required to observe synergisticinhibition of viral entry. sCD4 demonstrated no enhancing or inhibitoryeffect on the primary isolate (FIG. 4B). In contrast, D1D2-Igαtpdramatically inhibited replication of both Bal and the primary isolate.Of note, D1D2-Igαtp fully suppressed 17b-mediated enhancement of bothBal and the primary isolate.

Example 6 Stoichiometry of gp120s D1D2-Igαtp Binding

To better understand why D1D2-Igαtp fails to enhance viral replicationtwo biochemical properties of this recombinant protein werecharacterized. Initially, it was asked how many gp120s could be loadedonto a single D1D2-Igαtp. In addition, the kinetics of theseinteractions was examined. D1D2-Igαtp, once assembled into an oligomer,should theoretically present twelve independent gp120 binding sites.However, it is unclear whether steric constraints would limit the numberof gp120s that actually bind at any given point in time.

To address this issue, a biosensor assay was established that wouldmeasure the ratio of gp120 to D1D2-Igαtp under conditions in which thenumber of gp120s bound to D1D2-Igαtp approached equilibrium. Protein-Gwas covalently coupled to a biosensor chip, which was subsequentlyloaded with fixed concentrations of D1D2-Igαtp. To this surfaceincreasing concentrations of gp120 were then added. Once the level ofgp120 approaches equilibrium, the number of gp120s bound per D1D2-Igαtpcan be determined by employing a standard calculation that relatesBiacore response units (RUs) to the mass of protein bound (see Example1).

Using a sensor chip loaded with 270 picograms (pg) of D1D2-Igαtp,concentrations of gp120 above 1800 nM approached equilibrium (FIG. 5A).From these curves the number of gp120s recognized by a single D1D2-Igαtpwere derived (FIG. 5B). Under the conditions employed, D1D2-Igαtp boundten gp120s simultaneously. Practical limitations, including injectionvolumes, protein concentration and a very slow apparent off-rate ofgp120 from D1D2-Igαtp, allowed the establishment of conditions atequilibrium was approached, but did not actually achieved. Therefore,the 10:1 ratio should be regarded as a minimum number of gp120s boundper D1D2-Igαtp. Additionally, because gp120s can vary up to 30 kDa insize, this ratio may change when different envelopes are employed.Nevertheless, this analysis demonstrates that D1D2-Igαtp can bind manygp120s simultaneously.

Example 7 Binding Kinetics

One of the most important properties of the CD4 fusion polypeptidesdisclosed herein protein is the enhanced avidity of these CD4 fusionpolypeptides for the CD4 receptor. For example, for a the CD4 fusionpolypeptides disclosed herein, twelve binding sites are included (asopposed to four binding sites). Thus, the avidity is greatly enhancedrelative to monomeric CD4 or a dimer or tetramer.

It was next asked whether differences in the binding kinetics ofD1D2-Igαtp versus monomeric sCD4 might help explain the difference inactivity of these two inhibitors at low concentrations. Either sCD4 orD1D2-Igαtp was coupled to a biosensor chip and the binding properties offour different envelope proteins were compared. The four gp120s weemployed were: 92MW959, an R5 specific clade C envelope; Th14-12, an R5specific clade B envelope; 92Ug21-9, an X4 specific lade A envelope; andNL4-3, an X4 specific clade B envelope. With the exception of NL-4-3,each of these envelopes was cloned after minimal passage in vitro (Gaoet al., J Virol 70, No. 3:1651, 1996).

A dramatic difference was noted in the manner in which all of theseenvelopes dissociated from D1D2-Igαtp relative to monomeric sCD4. FIG.6A displays the dissociation curves of each of the envelopes from eitherD1D2-Igαtp or sCD4. The rate of dissociation is reflected in the slopeof the curve such that the more negative the slope the faster the rateof dissociation, while a slope of zero reflects constitutive binding. Itis clear from each of the dissociation curves that all of the envelopesdissociate more slowly from D1D2-Igαtp than from sCD4. Of note, each ofthese curves of D1D2-Igαtp dissociating from gp120 approaches a slopeclose to zero. These observations are most easily explained by assumingthat once an envelope dissociates from one chain of D1D2-Igαtp, itimmediately rebinds to the same molecule. Under conditions where thistype of rebinding is likely to occur it was not possible to calculate anaccurate dissociation constant (k_(d)). Nevertheless, by comparing thesCD4 and D1D2-Igαtp dissociation curves it was concluded that gp120dissociates from D1D2-Igαtp at a much slower rate than it dissociatesfrom monomeric sCD4.

The binding rate of D1D2-Igαtp for soluble monomeric gp120 employed inthis assay are likely to be different than those for gp120 presented onthe surface of an infectious virion. The CD4 binding epitope on gp120 isoccluded when gp120 is incorporated into a spike (Stamatatos et al., JVirol 69, No. 10:6191, 1995) thus reducing its accessibility, (i.e. rateof association), to both membrane bound CD4 as well as sCD4. However,the virion as a target presents on average 216 gp120s distributed astrimmers among 72 spikes (Ozel et al., Arch Virol 100, No.3-4:255,1988). To the extent that D1D2-Igαtp may bind more than one gp120simultaneously, the rate of dissociation from the virion should be evenslower than from a monomeric gp120. The data indicates that, relative tomonomeric sCD4, the multivalent nature of D1D2-Igαtp results in slowrates of dissociation from gp120 in a manner that makes D1D2-Igαtp amore efficient inhibitor of viral entry. To determine if D1D2-Igαtpdemonstrated high avidity toward HIV gp120 we linked gp120 to the sensorchip at high density and passed increasing concentrations of D1D2-Igαtpover the surface (FIG. 6B). The rate of dissociation is reflected by theslope of the dissociation phase. As can be observed, the slope is closeto zero, indicating an extremely avid interaction between HIV gp120 andD1D2-Igαtp.

Example 8 Size and Molar Mass Distribution of D1D2-Igαtp

Initially it was postulated that if D1D2-Igαtp were sufficiently largeit would prevent the enhancement of viral entry that is associated withsuboptimal concentrations of monomeric sCD4. Additionally, establishingthe size of D1D2-Igαtp would further help determine if it issufficiently large to span multiple spikes on the surface of a virion.Protein was initially fractionated by gel filtration and the peakfraction and trailing fraction were collected. Because of the well-knowndifficulty of precisely measuring the molar mass of large glycoproteinsby gel filtration, the size of D1D2-Igαtp was characterized in moredetail by analytical ultracentrifugation and dynamic light scattering.The homogeneity of the peak protein fraction was assessed bysedimentation velocity, which showed a broad sedimentation coefficientdistribution indicating a heterogeneous size distribution. The largemajority of protein in the peak fraction exhibited a sedimentationcoefficient between 14 and 25 S (FIG. 7, solid line). Consistent withthis observed heterogeneity, the average molar mass measured wasdependent on rotor speed, ranging from 5.8 to 8.8 monomer units (FIG. 7,top inset). In order to simplify the analysis of the size-distribution,the trailing fraction which exhibited less heterogeneity was alsostudied (FIG. 7, dashed line). By comparing the shape of both curves therange of sedimentation coefficients for each oligomer were estimated(arrows in FIG. 7). From these estimations the hydrodynamic radius ofthe pentamers up to octamers were calculated. The majority of themolecules was calculated to be 11.9-13.5 nm (FIG. 7). This was inexcellent agreement with a direct measurement of the hydrodynamic radiusby dynamic light scattering, which resulted in a peak at 12.5 nm for thetrailing fraction, and significant scattering from the larger oligomerscontained in the peak fraction (FIG. 7, bottom inset).

Although the hydrodynamic radius by itself does not contain informationabout the precise shape of the molecules, for fundamental reasons atleast in one dimension the molecules will measure at least twice thehydrodynamic radius. Therefore, it was concluded that D1D2-Igαtppreparation consists of molecules that are at least 24 nm in length.Given that a spike protrudes 10 nm from the surface of a virion(Gelderblom et al., Virology 156, No. 1:171, 1987) it can be consideredthat, once engaged by D1D2-Igαtp, spikes are impeded from interactingwith the target cell membrane. Furthermore, the distance from the centerand edge of one virion spike to an adjacent spike are 22 nm and 8 nmrespectively (Ozel et al., Arch Virol 100, No. 3-4:255, 1988; Gelderblomet al., Virology 156, No. 1:171, 1987; Forster et al., J Mol Biol 298,No. 5:841, 2000; Poignard et al., Annu Rev Immunol 19:253, 2001). Thusthe data indicates that a D1D2-Igαtp spans multiple spikes on the virionmembrane.

Thus, by increasing both the size and the valency of sCD4 one cangenerate a novel protein that is a highly potent inhibitor ofreplication and that, unlike monomeric sCD4, does not enhance virusreplication at suboptimal concentrations. This has importantimplications for therapeutic and vaccine strategies. Unlike coreceptorepitopes on gp120, the CD4 receptor binding site is highly conservedmaking it an attractive target for antiviral therapy. Theseconsiderations led to an examination of the biological attributes ofsCD4 that prevents it from inhibiting viral replication of primaryisolates.

The enhancing activity of sCD4 on HIV entry is considered to be one ofthe critical unintended effects of this potential anti-viral agent thathas led to its failure in clinical trials. As disclosed herein, byincreasing both the size and the valency of sCD4 an agent can begenerated that no longer enhances viral replication at sub-optimalconcentrations.

An extremely large immunoglobulin derivative, termed D1D2-Igαtp, hasbeen constructed that is comprised of, on average, twelve IgG1 heavychains fused to the two amino-terminal domains of CD4. The CD4 receptoris thought to extend about 7 nm from the membrane of a lymphocyte, whilethe extracellular loops of CCR5 lie closer to the cell surface (Poignardet al., Annu Rev Immunol 19:253, 2001). One of the proposed functions ofmembrane-bound CD4 is to bring the virion into close proximity to CCR5and thus to the cell membrane so that the fusion process can proceed.This process is dependent in part on CD4-induced conformational changesin gp120 (Doranz et al., J Virol 73, No. 12:10346, 1999; Trkola et al.,Nature 384, No. 6605:184, 1996; Wu et al., Nature 384, No. 6605:179,1996; Zhang et al., Biochemistry 38, No. 29:9405, 1999). Thus, withoutbeing bound by theory, by generating a molecule of sufficient size, suchas the D1D2-Igαtp, the attachment of such agents to the surface of avirion prevents that virion from gaining close proximity to fusioncomponents on the cell surface.

In this instance, any conformational changes in gp120 induced by such anagent are less likely to promote fusion. Viral spikes are estimated torise 10 nm from the surface of a virion (Ozel et al., Arch Virol 100,No. 3-4:255, 1988; Gelderblom et al., Virology 156, No. 1:171, 1987).The data from dynamic light-scattering experiments and sedimentationvelocity centrifugation indicate that the hydrodynamic radius ofD1D2-Igαtp is approximately 12 nm (diameter=24 nm). Thus, without beingbound by theory, it is likely that once D1D2-Igαtp engages a spike andinduces a conformational change in the envelope, that spike is unlikelyto gain the close proximity to the target cell membrane necessary forfusion to occur because of the bulk of the hexameric D1D2-Igαtp.

In viral entry assays D1D2-Igαtp inhibited viral entry at very lowconcentrations relative to monomeric sCD4. Furthermore, unlike sCD4,there was never an observation of any significant enhancing activity asthe D1D2-Igαtp was titered out. This is consistent with the hypothesisthat a large molecule renders induction of fusion components on thevirion irrelevant.

Unlike monomeric sCD4, D1D2-Igαtp inhibited primary isolates atrelatively low concentrations. This activity could result in part fromthe presentation by D1D2-Igαtp of twelve gp120 binding sites in closeproximity to each other. By presenting CD4 as a soluble dodecamer it canmore effectively compete with clusters of CD4 receptors on the targetcell membrane. When the dissociation of gp120 from monomeric sCD4 andD1D2-Igαtp were compared it was found that gp120 dissociated much moreslowly from D1D2-Igαtp than sCD4. The rate of dissociation of D1D2-Igαtpfrom soluble gp120 is likely to be different from that ofvirion-associated gp120. However, it is also likely that the trend isthe same: D1D2-Igαtp also likely dissociates from virion spikes moreslowly than monomeric sCD4.

Without being bound by theory, it is possible that a single D1D2-Igαtpbinds more than one of the three gp120s included in a spike. It wasdetermined that a single D1D2-Igαtp is either sufficiently flexible orotherwise folded to accommodate at least 10 gp120s, supporting thepossibility that two or even three of the envelopes on a spike could beoccupied by different chains of a single D1D2-Igαtp. Additionally,because spikes on a virion are arranged approximately 22 nm apart(center to center), a single D1D2-Igαtp, with an estimated diameter of24 nm, may span multiple spikes. Binding of one D1D2-Igαtp to multipleenvelopes on a virion, whether within or across spikes, shouldsignificantly slow the rate at which it dissociates from that virion. Tothe extent that spikes are occupied and kept sufficiently distant fromthe cell membrane they cannot participate in the fusion process. Thusthe size and capacity for multivalent ligation confer upon D1D2-Igαtptwo properties that distinguish it from monomeric sCD4: (1) it does notenhance viral replication at suboptimal concentrations, and (2) itefficiently inhibits replication of primary isolates. These propertiesconfer on D1D2-Igαtp therapeutic properties not observed with monomericsCD4 in previously reported clinical trials (Schooley et al., AnnIntern. Med 112, No.4:247, 1990).

Similar to sCD4, mAbs specific for gp120 car enhance the replication ofmany primary isolates. Additionally, polyclonal sera from infectedpatients, or individuals vaccinated with envelope based immunogens alsoenhance HIV-1 replication (Sullivan et al., J Virol 69, No. 7:4413,1995; Sorensen et al., J Immunol 162, No. 6:3448, 1999). As with sCD4,this effect is seen as the concentration of the sera or mAb is titeredout to very high dilutions. As has been noted elsewhere, this propertyof gp120-specific antibodies may negatively impact on the effectivenessof anti-envelope humoral responses, both in the context HIV disease aswell as vaccination. In order to ask whether D1D2-Igαtp would interferewith antibody-mediated enhancement of viral replication mAb 17b, anantibody that has previously been shown to enhance viral replication,was employed. Of note, 17b recognizes gp120 more efficiently in thepresence of monomeric sCD4 (Sullivan. et al., J Virol 69, No. 7:4413,1995; Schutten et al., Scand J Immunol 41, No. 1:18, 1995; Doranz etal., J Virol 73, No. 12:10346, 1999). In fact, D1D2-Igαtp eliminated theenhancing effects of 17b. Without being bound by theory, this may haveoccurred because the size of D1D2-Igαtp limits the access of 17b to thevirion, or it may have prevented 17b enhancement by keeping virionspikes at a distance from the target cell membrane (see above). In thisrespect, D1D2-Igαtp illustrates two highly desirable attributes of apotent neutralizing antibody, it dissociates slowly from gp120, and itis large and therefore likely to keep the virion separated from the cellmembrane.

In summary, disclosed herein are CD4 fusion proteins that address one ofthe principle properties of sCD4 that has prevented sCD4 from beingdeveloped as an effective antiviral agent. Since sCD4 binds to one ofthe few structures on gp120 that is almost invariably conserved inreplication competent viruses, the CD4 binding epitope on gp120 remainsa highly attractive target for both therapeutic strategies and vaccines.Disclosed herein are molecules that do not have the intrinsic capacityof sCD4 to enhance viral replication, but have an increased ability tosuppress virus replication. Thus, this disclosure provides novel insightinto requirements of effective inhibitors of viral entry, and providesmolecules that are of use as therapeutic modalities.

Example 9 D1D2-Igαtp Neutralizes CCR5-Utilizing Primary Isolates

The ability of D1D2-Igαtp to neutralize four primary isolates of HIV-1was tested. In addition, two viruses, Bal and JR-F1 derived frommolecularly cloned HIV viruses were also tested. A standard virusneutralization assay (Letvin et al., J. Virol 75:4165-4175, 2001) inwhich decreasing concentrations of D1D2-Igαtp were incubated with theeach of the six isolates and activated peripheral blood mononuclearcells. Parallel experiments were carried out with sCD4 as a point ofcomparison. Twenty-four hours post inoculation cells were washed andplaced into standard culture conditions. Supernatants were collectedover a period of fourteen days. Viral replication was assessed bymeasurement of reverse transcriptase in culture supernatants. The datais shown in FIG. 8, wherein the data presented represents the percentinhibition on a day prior to the peak day of viral replication relativeto a parallel culture in which no inhibitor was added. The 90%inhibitory concentrations of D1D2-Igαtp are as good or better than themost efficient neutralizing antibodies (Burton et al., Science 2661024-1027, 1994).

D1D2-Igαtp inhibited viral replication in each primary viral isolate by90% at a concentration below 2.8 nM (see FIG. 8).

Example 10 Inhibition of Viral Entry After Attachment of the Virus tothe Target Cell Membrane

A viral entry assay, taqman (see Example 3), was used to determine theability of D1D2-Igαtp to inhibit viral entry. Briefly, PBMCs wereexposed to virus (HIV-1 Bal) for one or two hours, and then washedextensively. Thus, the only virus remaining is that already attached tothe surface of cells. D1D2-Igαtp was the added to the cells at aconcentration of 25 nM. Sixteen hours later, the cells were lysed and ataqman based assay was performed. As shown in FIG. 9, D1D2-Igαtpinhibited the entry of virus, subsequent to initial attachment of thevirions to cells into CD4+ T-cells by 50%. Without being bound bytheory, it is possible that this result reflects inactivation of virusbound to cells through proteoglycans prior to specific interaction ofthe gp120 component with CD4.

Example 11 Generation of a Second D1D2-Igαtp with Altered Properties

As disclosed above, the DNA coding sequence of D1D2-Igαtp is:

ATGAACCGGGGAGTCCCTTTTAGGCACTTGCTTCTGGTGCTGCAACTGGC (SEQ ID NO:5)GCTCCTCCCAGCAGCCACTCAGGGAAAGAAAGTGGTGCTGGGCAAAAAAGGGGATACAGTGGAACTGACCTGTACAGCTTCCCAGAAGAAGAGCATACAATTCCACTGGAAAAACTCCAACCAGATAAAGATTCTGGGAAATCAGGGCTCCTTCTTAACTAAAGGTCCATCCAAGCTGAATGATCGCGCTGACTCAAGAAGAAGCCTTTGGGACCAAGGAAACTTCCCCCTGATCATCAAGAATCTTAAGATAGAAGACTCAGATACTTACATCTGTGAAGTGGAGGACCAGAAGGAGGAGGTGCAATTGCTAGTGTTCGGATTGACTGCCAACTCTGACACCCACCTGCTTCAGGGGCAGAGCCTGACCCTGACCTTGGAGAGGCCCCCTGGTAGTAGCCCCTCAGTGCAATGTAGGAGTCCAAGGGGTAAAAACATACAGGGGGGGAAGACCCTCTCCGTGTCTCAGCTGGAGCTCCAGGATAGTGGCACCTGGACATGCACTGTCTTGCAGAACCAGAAGAAGGTGGAGTTCAAAATAGACATCGTGGTGCTAGCTTCGGCCGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGGGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAACCAGGTCAGGCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTGTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTAAGCTTGTCTGCGGGTAAACCCACCCATGTCAATGTGTCTGTTGTCATGGCGGAGGTGGACGGCACCTGCTAC TGA

The amino acid sequence of D1D2-Igαtp is:

MNRGVPFRHLLLVLQLALLPAATQGKKVVLGKKGDTVELTCTASQKKSIQF (SEQ ID NO:6)HWKNSNQIKILGNQGSFLTKGPSKLNDRADSRRSLWDQGNFPLIIKNLKIEDSDTYICEVEDQKEEVQLLVFGLTANSDTHLLQGQSLTLTLESPPGSSPSVQCRSPRGKNIQGGKTLSVSQLELQDSGTWTCTVLQNQKKVEFKTDIVVLASADKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSAGKPTHVNVSVVMAEVDGTCY*.

Using molecular cloning procedures and site directed mutagenesis of DNAas has been previously described (see Kim and Maas, Biotechniques28(2):196-8, 2000), a variant (termed FD1D2-Igαtp, or mutant F) wasproduced. This variant includes amino acids of an IgG₂ that are involvedin binding to Fc receptors. The DNA coding sequence of FD1D2-Igαtp is asfollows:

ATGAACCGGGGAGTCCCTTTTAGGCACTTGCTTCTGGTGCTGCAACTGGC (SEQ ID NO:10)GCTCCTCCCAGCAGCCACTCAGGGAAAGAAGTGGTGCTGGGCAAAAAAGGGGATACAGTGGAACTGACCTGTACAGCTTCCCAGAAGAAGAGCATACAATTCCACTGGAAAAACTCCAACCAGATAAAGATTCTGGGAAATCAGGGCTCCTTCTTAACTAAAGGTCCATCCAAGCTGAATGATCGCGCTGACTCAAGAAGAAGCCTTTGGGACCAAGGAACTTCCCCCTGATCATCAAGAATCTTAAGATAGAAGACTCAGATACTTACATCTGTGAAGTGGAGGACCAGAAGGAGGAGGTGCAATTGCTAGTGTTCGGATTGACTGCCAACTCTGACACCCACCTGCTTCAGGGGCAGAGCCTGACCCTGACCTTGGAGAGCCCCCCTGGTAGTAGCCCCTCAGTGCAATGTAGGAGTCCAAGGGGTAAAAACATACAGGGGGGGAACACCCTCTCCGTGTCTCAGCTGGAGCTCCAGGATAGTGGCACCTGGACATGCACTGTCTTGCAGAACCAGAAGAAGGTGGAGTTCAAAATAGACATCGTGGTGCTAGCTTCGGCCGACAAAACTCACACATGCCCACCGTGCCCAGCACCTCCAGTCGCGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGGGTGGTCAGCGTCCTCACCGTCCTGCACCAGGAGTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGGCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTAAGCTTGTCTGCGGGTAAACCCACCCATGTCAATGTGTCTGTTGTCATGGCGGAGGTGGACGGCACCTGCTACTGA

This nucleic acid sequence encodes the following amino acid sequence:

MNRGVPFRHLLLVLQLALLPAATQGKKVVLGKKGDTVELTCTASQKKSIQF (SEQ ID NO:11)HWKNSNQIKILGNQGSFLTKGPSKLNDRADSRRSLWDQGNFPLIIKNLKIEDSDTYICEVEDQKEEVQLLVFGLTANSDTHLLQGQSLTLTLESPPGSSPSVQCRSPRGKNIQGGKTLSVSQLELQDSGTWTCTVLQNQKKVEFIKDIVVLASADKTHTCPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSAGKPTHVNVSVVMAEVDGTCY,termed FD1D2-Igαtp.

The differences between the D1D2-Igαtp (SEQ ID NO: 5) and FD1D2-Igαtp(SEQ ID NO: 10) nucleic acid sequences are as follows:

-   -   1. nucleotide 652 of D1D2-Igαtp was changed from G to C    -   2. nucleotide 653 of D1D2-Igαtp was changed from A to C    -   3. nucleotide 655 of D1D2-Igαtp was changed from C to G    -   4. nucleotides 658-660 of D1D2-Igαtp were deleted    -   5. nucleotide 662 of D1D2-Igαtp was changed from G to C

The resulting differences between the D1D2-Igαtp (SEQ ID NO: 6) andFD1D2-Igαtp (SEQ ID NO: 11) are in amino acid sequences 218-221 (see SEQID NO:6). These substitutions are as follows (see FIG. 14 for referencepoints on an intact IgG, residues 218-221 in D1D2-Igαtp correspond toresidues 233-238 in an intact IgG₁):

-   -   D1D2-Igαtp: Glu Leu Leu Gly    -   FD1D2-Igαtp: Pro Val—Ala

IgG₂s exhibit a substantially lower affinity for CD16, CD32 and CD64when compared to standard human IgG₁s. This difference occurs as aconsequence of different amino acids in the IgG₁ and IgG₂ at specificpositions in the proteins that interact directly with CD16, CD32 andCD64 (see FIG. 14). These amino acids and their role in the recognitionof Fc receptors have been described extensively (see Shields et al., J.Biol Chem. 276(9):6591-604, 2001). The residues in D1D2-Igαtp (shownabove) contain substitutions such that the residues involved in IgG₁recognition were replaced with the corresponding residues encoded byIgG₂ (FIG. 15). Thus, a chimeric IgG1/IgG2 CH2CH3 domain has beenincluded within the overall framework of D1D2-Igαtp. The resultingmolecule, FD1D2-Igαtp, binds to CD16 and Cd32 with a substantially lowerapparent affinity, as described below.

Example 12 FD1D2-Igαtp Binds to Fc Receptors with an Altered Affinity

As disclosed herein, D1D2-Igαtp is a fusion protein made from threeproteins: human CD4, human IgG1, and human IgA. The domains of humanIgG₁ that are included in D1D2Igαtp are termed “CH2CH3.” The CH2CH3domain includes a specific epitope that binds to a family of receptorscalled the Fc receptors. The three most extensively characterized Fcreceptors are termed CD16, CD32, and CD64. A standard human IgG1antibody binds to CD16 and CD32 with low affinity.

As a consequence of the extensive polymerization of CH2CH3 in thecontext of D1D2-Igαtp there is a high affinity of this molecule of CD16and CD32 receptors (see FIG. 10). In order to compare the bindingaffinity of D1D2-Igαtp and FD1D2-Igαtp competition experiments for CD16binding were performed using a fluorescence labeled anti CD16 antibodyas a competitor. Cells were incubated at 4° C. with a constant amount ofthe labeled anti CD16 antibody and increasing concentrations ofD1D2-Igαtp or FD1D2-Igαtp. The extent of anti CD16 binding was measuredby flow cytometry. The % CD16 mean channel fluorescence (mcf) wascalculated as follows:

${\%\mspace{14mu}{CD16}\mspace{14mu}{mcf}} = {\frac{\begin{matrix}{\left( {{CD16mcf}\text{-}{backgroud}} \right) -} \\\left( {{CD16}\mspace{14mu}{with}\mspace{14mu}{inhibitor}\mspace{14mu}{mcf}\text{-}{background}} \right.\end{matrix}}{\left( {{CD16}\mspace{14mu}{mcf}\text{-}{background}} \right)} \times 100}$

The results demonstrate that D1D2-Igαtp efficiently competes for bindingto CD16, while FD1D2-Igαtp competes less efficiently (FIG. 10A).Antibody 2G12 (negative control, a human IgG₁), did not compete forbinding to CD16.

In order to compare the binding affinity of D1D2-Igαtp and FD1D2-Igαtp,competition experiments for CD32 binding were also performed. Thesestudies used a labeled anti-CD32 antibody as a competitor. The % CD32mean channel fluorescence (mcf) was calculated as follows:

${\%\mspace{14mu}{CD32}\mspace{14mu}{mcf}} = {\frac{\begin{matrix}{\left( {{CD32mcf}\text{-}{backgroud}} \right) -} \\\left( {{CD32}\mspace{14mu}{with}\mspace{14mu}{inhibitor}\mspace{14mu}{mcf}\text{-}{background}} \right.\end{matrix}}{\left( {{CD32}\mspace{14mu}{mcf}\text{-}{background}} \right)} \times 100}$

FIG. 10B shows the binding to CD32 obtained in the presence of 1-1000 nMof competitor (2G12, a human IgG₁). D1D2-Igαtp efficiently competes forbinding to CD32, while FD1D2-Igαtp competes less efficiently. Antibody2G12, (negative control, a human IgG₁), did not compete for binding toCD32.

Example 13 Effect of FD1D2-Igαtp and D1D2-Igαtp on Natural Killer Cells

The binding and cross-linking of CD16 on antigen presenting cells andnatural killer (NK) cells by IgG₁s results in biological responses inthose cells that promote immune responses. In one example, this responsecan be measured by measuring a calcium flux in NK cells. Calcium influxcan be measured as described in Rabin et al., J Immunol. 162(7):3840-50,1999.

Representative plots showing the induction of a calcium flux byD1D2-Igαtp or FD1D2-Igαtp (mutant F) in natural killer (NK) cells afterthe cells were cultured in vitro for 14 days are shown in FIG. 11. Thenegative control (SHAM, FIG. 11A) did not exhibit any calcium influx,while application of different concentrations of D1D2-Igαtp (FIGS.11B-F, 120 nm, 60 nm, 30 nm, 15 nM, and 7.5 nM, respectively) elicited acalcium influx. Mutant F application did not induce a calcium influx(FIG. 11GK, 120 nm, 60 nm, 30 nm, 15 nM, and 7.5 nM, respectively),indicated that this molecule does not activate natural killer cells.

The results demonstrate that as D1D2-Igαtp has a high affinity for theFc receptor, there is resulting enhanced signal transduction through theCD16 receptor on human primary natural killer (NK) cells. This signaltransduction induced by D1D2-Igαtp in NK cells that are of asubstantially greater magnitude than signals delivered by human IgG1antibodies (See FIG. 11).

Example 14 D1D2-Igαtp Mediates Cytoxicity of HIV Infected Cells

Fluorescence activated cell sorting analyses were performed todemonstrate that D1D2-Igαtp mediates antibody dependent cell mediatedcytoxicity. HIV-infected CEM.NRK target cells were incubated in thepresence of NK cells with either D1D2-Igαtp or in media alone. Cellswere subsequently labeled with propidium iodide, which measures cellviability (viable cells exclude propidium iodide).

In the presence of D1D2-Igαtp, 45% of the HIV-1 infected cells werekilled by the NK cells, whereas without application of D1D2-Igαtp, only15% of the cells were killed. The same number of uninfected CEM.NRKcells survived in the presence of D1D2-Igαtp as compared to uninfectedCEM.NRK cells incubated in the absence of antibody (FIG. 12). Thus,D1D2-Igαtp mediates NK cell mediated antibody dependent cell mediatedcytoxicity. The percent of PI positive cells obtained after incubationof HIV-infected CEM.NRK target cells with either D1D2-Igαtp or mutant F(FD1D2-Igαtp) was compared (FIG. 13). Both D1D2-Igαtp and mutant Finduced killing, although D1D2-Igαtp was more effective.

In view of the many possible embodiments to which the principles of thisdisclosure may be applied, it should be recognized that the illustratedembodiment is only a preferred example of the invention and should notbe taken as a limitation on the scope of the invention. Rather, thescope of the invention is defined by the following claims. We thereforeclaim as our invention all that comes within the scope and spirit ofthese claims.

1. A recombinant polypeptide comprising a CD4 polypeptide ligated at itsC-terminus with an immunoglobulin polypeptide, wherein theimmunoglobulin polypeptide comprises a hinge region and a constantdomain of a mammalian immunoglobulin heavy chain, and wherein theimmunoglobulin polypeptide is fused at its C-terminus with a tailpiecepolypeptide from the C terminus of the heavy chain of an IgA antibody ora tailpiece from a C terminus of the heavy chain of an IgM antibody. 2.The recombinant polypeptide of claim 1, wherein the CD4 polypeptidecomprises the D1 and D2 regions of a human CD4 polypeptide.
 3. Therecombinant polypeptide of claim 1, wherein the immunoglobulinpolypeptide is a human IgG molecule.
 4. The recombinant polypeptide ofclaim 3, wherein the imrnunoglobulin polypeptide is an IgG₁, IgG₂ or anIgG₃, or a combination thereof.
 5. The recombinant polypeptide of claim4, wherein the tailpiece polypeptide comprises the tailpiece from the Cterminus of the heavy chain of an IgA antibody.
 6. The recombinantpolypeptide of claim 1, wherein the constant domain comprises a CH2domain.
 7. The recombinant polypeptide of claim 6, wherein the constantdomain further comprises a CH3 domain.
 8. The recombinant polypeptide ofclaim 1, comprising a polypeptide linker between the CD4 polypeptide andthe inununoglobulin polypeptide.
 9. A composition comprising therecombinant polypeptide of claim 1 in a carrier.
 10. A kit for treatmentor prevention of HIV infection, comprising a container comprising aneffective amount of the recombinant polypeptide of claim
 1. 11. Amultimeric polypeptide comprising monomers of the recombinantpolypeptide of claim
 5. 12. The multimeric polypeptide of claim 11,wherein the multimer is a dodecylmer.
 13. The recombinant polypeptide ofclaim 5, comprising SEQ ID NO:
 1. 14. The recombinant polypeptide ofclaim 13, wherein the immunoglobulin is an IgG₁.
 15. The recombinantpolypeptide of claim 13, wherein the polypeptide comprises SEQ ID NO: 6or SEQ ID NO:
 11. 16. The recombinant polypeptide of claim 13, whereinthe polypeptide consists of SEQ ID NO: 6 or SEQ ID NO: 11.